Method for obtaining polypeptide constructs comprising two or more single domain antibodies

ABSTRACT

The present invention relates to methods for obtaining a polypeptide construct directed against one or more antigens and/or epitopes and having one or more desired characteristics, wherein the polypeptide construct comprises at least two single domain antibodies. The methods of the present invention involve producing a diversity of polypeptide constructs that are structural variants and screening the produced diversity of polypeptide constructs for a polypeptide construct having said one or more desired characteristics. The present invention further relates to polypeptide constructs directed against one or more antigens and/or epitopes having one or more desired characteristics, wherein the polypeptide construct comprises at least two single domain antibodies. The methods and polypeptide constructs according to the present invention are useful for the identification of optimal therapeutic compounds.

FIELD OF THE INVENTION

The present invention relates to the area of antibody engineering and inparticular concerns a method for obtaining polypeptide constructsdirected against one or more antigens and/or epitopes, said polypeptideconstructs having one or more desired characteristics.

BACKGROUND OF THE INVENTION

Antibody constructs comprising more than one binding site and/or bindingunit are known to have several advantages compared to antibodies andantibody fragments comprising only one single binding site, such as animproved potency, bi- or multispecificity, multifunctionality, etc.

Typically, individual binding units are first isolated and extensivelycharacterized, prior to composing an antibody construct combining thesebinding units. The method of linking the binding units by means oflinker sequences, the composition of these linker sequences and theorientation and order of the binding units, as well as the choice of thebinding unit combination itself all can affect the specificcharacteristics of the antibody construct, such as for example theaffinity and/or avidity for one or more antigens, the expression levelsand/or the stability of the antibody construct, etc. For instance,although some binding units may function very well individually, theirbinding behavior may be impaired upon linkage to another binding unitand/or component. Alternatively, some binding units may performsuboptimally when tested in their individual form but could neverthelessmake suitable linkage partners in the context of a particular antibodyconstruct, e.g. their characteristics may be complementary to those ofother binding units and/or components.

McGuinness et al. (Nature Biotechnology 14: 1149-1154 (1996)) developeda method that allows the generation and screening of repertoires ofbispecific antibody constructs, called “diabodies”. Although thesediabody libraries allow screening for the optimal bispecific moleculewith regard to particularly desired properties, such as binding affinityor epitope recognition, this known production method is hampered by itscomplex and laborious cloning procedures.

The published international applications WO 03/002609, WO 04/003019, WO04/058821 and WO 08/096,158 disclose methods for producing dual-specificligands by screening libraries of heavy and light chain variable domainsderived from conventional four chain antibodies for a particular heavychain variable domain and a particular light chain variable domain andsubsequently combining these to form a dual-specific ligand. It isfurthermore described in these applications that optionally libraries ofstructural and/or functional variants of the obtained dual-specificligand can be produced in order to select the most optimal dual-specificligand.

However, the potential of the methods described in the prior art islimited since only libraries composed of bivalent and/or bispecificfragments can be produced (i.e. precluding the possibility to producelibraries composed of more complex antibody constructs with multivalencyand/or multispecificity). In addition, the format of antibody constructspresent in the libraries of the prior art is less suitable for theproduction of biparatopic molecules (i.e. antibody constructs comprisingat least two binding units that bind to two different epitopes on thesame antigen). Finally, it is noted that the antibody constructlibraries of the prior art are produced at random and therefore arecharacterized by the presence of quite a lot of unfavorablecombinations, which hampers screening for the optimal bispecificmolecule.

SUMMARY OF THE INVENTION

The present invention provides rapid and efficient methods for obtaininga polypeptide construct, which methods overcome the drawbacks andlimitations of the methods described in the prior art. By using thesmallest antigen binding antibody fragments (i.e. single domainantibodies) as basic building blocks for the production of polypeptideconstructs, the methods of the invention allow to easily and rapidlyprepare and screen large numbers of multivalent, multispecific and/ormultiparatopic polypeptide constructs in order to obtain a particularpolypeptide construct having specific desired characteristics. Thesystem bypasses the need to first extensively characterize theindividual binding units, by testing the performance of these bindingunits directly in the context of a particular polypeptide construct,potentially revealing functional features not exhibited by theindividual binding units.

The invention provides methods wherein a template polypeptide constructis selected, a diversity of structural variants for the template aregenerated, and the diversity of constructs is screened for a polypeptideconstruct having one or more suitable characteristics, more particularlyhaving two or more suitable characteristics.

Thus, according to one aspect, the present invention relates to methodsfor obtaining a polypeptide construct having one or more desiredcharacteristics, wherein the polypeptide construct comprises at leasttwo single domain antibodies and is directed against one or moreantigens and/or epitopes, which methods at least comprise the steps of:

-   -   (i) selecting a template polypeptide construct    -   (ii) producing a diversity of polypeptide constructs that are        structural variants for the selected template polypeptide        construct of step (i), wherein said structural variants each        comprise at least two single domain antibodies, and    -   (iii) screening the produced diversity of polypeptide constructs        of step (ii) for a polypeptide construct having said one or more        desired characteristics, wherein said polypeptide construct        comprises at least two single domain antibodies and is directed        against one or more antigens and/or epitopes.

In particular embodiments, the polypeptide constructs obtained accordingto the methods of the invention comprising at least two single domainantibodies comprise at least two single domain antibodies selected fromdomain antibodies, “dAbs”, Nanobodies®, V_(HH) sequences and othersingle variable domains including combinations thereof. For instance,particular embodiments of the methods of the invention comprise theproduction of polypeptide constructs wherein the at least two singledomain antibodies are selected from V_(H) domains and/or V_(L) domains(both derived from conventional four-chain antibodies) and/or V_(HH)domains (derived from heavy chain antibodies). More particularembodiments of the invention relate to the production of polypeptideconstructs wherein the single domain antibodies consist essentially ofonly one type of domain antibody (i.e. corresponding to either heavy orlight chain domains). Most particularly it is envisaged that the methodsof the present invention involve the generation of polypeptideconstructs wherein the single domains exclusively consist of heavy chaindomain antibodies (e.g. V_(H) or V_(HH)). Further particular embodimentsof the invention involve methods wherein the polypeptide constructsgenerated comprise at least two single domains, whereby the singledomains of the construct consist exclusively of V_(HH) domains orexclusively consist of heavy chain variable domains derived from heavychain antibodies.

In a particular embodiment of the invention, the diversity ofpolypeptide constructs can be a set, collection or library ofpolypeptide constructs. More particularly, the diversity of polypeptideconstructs can be a library of polypeptide constructs.

Also, the diversity of polypeptide constructs may be a set, collectionor library of polypeptide constructs comprising at least two singledomain antibodies that are exclusively heavy chain variable domains(e.g. V_(H) or V_(HH)). More particularly, the diversity of polypeptideconstructs may be a set, collection or library of polypeptide constructscomprising at least two single domain antibodies that are exclusivelyheavy chain variable domains of heavy chain antibodies (V_(HH) domains).

In further particular embodiments, step (ii) of the methods of theinvention of producing a diversity of polypeptide constructs that arestructural variants for the selected template polypeptide construct ofstep (i), comprises producing one or more of the following:

-   -   structural variants with regard to the number and/or identity of        said single domain antibodies in said polypeptide constructs,        and/or,    -   structural variants with regard to the relative position of said        single domain antibodies within the polypeptide constructs,        and/or,    -   structural variants with regard to the amino acid residues of        the CDR regions of said single domain antibodies in said        polypeptide constructs, and/or,    -   structural variants with regard to the amino acid residues of        the framework regions of said single domain antibodies in said        polypeptide constructs, and/or,    -   structural variants with regard to the codon usage of said        selected template polypeptide construct.

It is envisaged that, in the methods of the invention the selectedtemplate polypeptide construct can a theoretical polypeptide construct,for which in the methods described herein a diversity of polypeptideconstructs are generated which are structural variants. This istypically the case when the starting point is only a desired feature(e.g. antigen binding). Alternatively, the selected polypeptideconstruct comprises a known (i.e. previously identified single domainantibody) and the diversity of polypeptide constructs comprisesstructural variants of the template polypeptide construct.

In particular embodiments, said step (ii) of the methods of theinvention of producing a diversity of polypeptide constructs that arestructural variants for the selected template polypeptide construct ofstep (i) comprises producing structural variants each comprising atleast two single domain antibodies that are linked via one or morepeptide linkers. In particular embodiments, modifying the peptidelinkers may favorably affect one or more desirable characteristics ofthe polypeptide construct.

Accordingly, in further particular embodiments of the methods describedherein, step (ii) of the methods of the invention of producing adiversity of polypeptide constructs that are structural variants for theselected template polypeptide construct of step (i), wherein saidstructural variants each comprise at least two single domain antibodiesthat are linked via one or more peptide linkers, comprises producing oneor more of the following

-   -   structural variants with regard to the composition of said one        or more peptide linkers, and/or,    -   structural variants with regard to the number of said one or        more linkers, and/or,    -   structural variants with regard to the relative position of said        one or more linkers in said polypeptide constructs.

Again, as described above, the template polypeptide construct may be atheoretical construct. Alternatively, the template polypeptide constructcomprises a known linker.

It will be understood to the skilled person that methods are equallyenvisaged wherein, upon generating a diversity of polypeptideconstructs, structural variants with regard to both the single variabledomains and the peptide linkers are also envisaged.

The methods of the present invention further comprise step (iii) ofscreening the diversity of polypeptide constructs produced in step (ii)for a polypeptide construct having one or more desired characteristics.In particular embodiments, the one or more desired characteristics maybe selected from characteristics such as (but not limited to) a suitablebinding affinity, a suitable solubility, a suitable stability, asuitable efficacy, and/or a suitable potency. It will be understood tothe skilled person that, in the selection of the template polypeptideconstruct, particular characteristics may be implied (such as binding tothe antigen of interest) and the screening step encompasses screeningfor one or more additional characteristics.

The methods of the present invention, involve the identification ofpolypeptide constructs which comprise at least two single domainantibodies. In particular embodiments, the methods envisage thegeneration of polypeptide constructs directed against one antigen. Inthese methods, the generated polypeptide constructs may comprise two ormore single domain antibodies directed against the same epitope ordirected against at least two different epitopes of the same antigen. Inyet further embodiments, the methods according to this embodiment mayinvolve the generation of polypeptide constructs comprising two or moresingle domain antibodies directed against different antigens. Inparticular embodiments of the methods described herein, the diversity ofpolypeptide constructs and the selected polypeptide construct comprisefor instance at least three single domain antibodies, wherein the atleast three single domain antibodies may be directed against the same orone or more (two or all three) different epitopes, which may be presenton the same antigen or which may be present on different antigens (suchas e.g. two single domain antibodies directed against the same epitopeand one single domain antibody directed against a different epitope onthe same antigen, two single domain antibodies directed against the sameepitope on a first antigen and one single domain antibody against adifferent antigen; two single domain antibodies directed against adifferent epitope on the first same antigen and one single domainantibody against a different antigen; three single domain antibodieseach directed against a different epitope on the same antigen; threesingle domain antibodies each directed against a different antigen).

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all terms used in disclosing the invention,including technical and scientific terms, have the meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. By means of further guidance, term definitions are included tobetter appreciate the teaching of the present invention. Reference isfor example made to the standard handbooks, such as Sambrook et al.,“Molecular Cloning: A Laboratory Manual” (2nd. Ed.), Vols. 1-3, ColdSpring Harbor Laboratory Press (1989); F. Ausubel et al, eds., “Currentprotocols in molecular biology”, Green Publishing and WileyInterscience, New York (1987); Lewin, “Genes II”, John Wiley & Sons, NewYork, N.Y., (1985); Old et al., “Principles of Gene Manipulation: AnIntroduction to Genetic Engineering”, 2nd edition, University ofCalifornia Press, Berkeley, Calif. (1981); Roitt et al., “Immunology”(6th. Ed.), Mosby/Elsevier, Edinburgh (2001); Roitt et al., Roitt'sEssential Immunology, 10^(th) Ed. Blackwell Publishing, UK (2001); andJaneway et al., “Immunobiology” (6th Ed.), Garland SciencePublishing/Churchill Livingstone, New York (2005), as well as to thegeneral background art cited herein.

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise.

The terms “comprising”, “comprises” and “comprised of” as used hereinare synonymous with “including”, “includes” or “containing”, “contains”,and are inclusive or open-ended and do not exclude additional,non-recited members, elements or method steps.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within the respective ranges, as well as the recitedendpoints.

With the term “polypeptide construct” as used herein is meant a compoundor polypeptide comprising one or more (preferably at least two) bindingunits that are linked to each other (optionally via linker sequencessuch as a peptide linker sequence) and/or optionally to other groups,residues, moieties or binding units (e.g. via disulphide bridges or vialinker sequences, such as peptide linker sequences). Preferably, saidone or more other groups, residues, moieties or binding units are aminoacid sequences.

In the context of the present invention, the polypeptide construct (alsoreferred to as “polypeptide construct of the invention”) comprises atleast two single domain antibodies (as defined herein), being directedagainst one or more antigens (as defined herein) and/or epitopes (asdefined herein). In the method of the present invention polypeptideconstructs are screened, selected and/or obtained that exhibit one ormore desired characteristics. The one or more desired characteristicsmay be (but are not limited to) a suitable binding affinity (asdescribed herein), a suitable solubility (as described herein), asuitable stability (as described herein), a suitable efficacy (asdescribed herein) and/or a suitable potency (as described herein). Apolypeptide construct of the invention with one or more desiredcharacteristics is obtained using the method of the present invention byscreening a diversity of polypeptide constructs for said one or moredesired characteristics.

The polypeptide construct of the invention comprising two or more singledomain antibodies are also be referred to herein as “multivalent”polypeptide constructs of the invention. Single domain antibodiespresent in such polypeptide constructs will also be referred to hereinas being in a “multivalent format”. For example a “bivalent” polypeptideconstruct of the invention comprises two single domain antibodies,optionally linked via a linker sequence, whereas a “trivalent”polypeptide of the invention comprises three single domain antibodies,optionally linked via two linker sequences; etc.;

In a multivalent polypeptide of the invention, the two or more singledomain antibodies may be the same or different, and may be directedagainst the same antigen or antigenic determinant (for example againstthe same part(s) or epitope(s) or against different parts or epitopes)or may alternatively be directed against different antigens or antigenicdeterminants; or any suitable combination thereof. For example, abivalent polypeptide construct of the invention may comprise (a) twoidentical single domain antibodies; (b) a first single domain antibodydirected against a first antigenic determinant of a protein or antigenand a second single domain antibody directed against the same antigenicdeterminant of said protein or antigen which is different from the firstsingle domain antibody; (c) a first single domain antibody directedagainst a first antigenic determinant of a protein or antigen and asecond single domain antibody directed against another antigenicdeterminant of said protein or antigen; or (d) a first single domainantibody directed against a first protein or antigen and a second singledomain antibody directed against a second protein or antigen (i.e.different from said first antigen). Similarly, a trivalent polypeptideconstruct of the invention may, for example and without being limitedthereto. comprise (a) three identical single domain antibody; (b) twoidentical single domain antibody against a first antigenic determinantof an antigen and a third single domain antibody directed against adifferent antigenic determinant of the same antigen; (c) two identicalsingle domain antibody against a first antigenic determinant of anantigen and a third single domain antibody directed against a secondantigen different from said first antigen; (d) a first single domainantibody directed against a first antigenic determinant of an antigen, asecond single domain antibody directed against a second antigenicdeterminant of said antigen and a third single domain antibody directedagainst a third antigenic determinant of the same antigen; (e) a firstsingle domain antibody directed against a first antigenic determinant ofa first antigen, a second single domain antibody directed against asecond antigenic determinant of said first antigen and a third singledomain antibody directed against a second antigen different from saidfirst antigen; or (f) a first single domain antibody directed against afirst antigen, a second single domain antibody directed against a secondantigen different from said first antigen, and a third single domainantibody directed against a third antigen different from said first andsecond antigen.

Polypeptide constructs of the invention that contain at least two singledomain antibody, in which at least one single domain antibody isdirected against a first antigenic determinant on an antigen and atleast one single domain antibody is directed against a second antigenicdeterminant on the same antigen will also be referred to as“multiparatopic” polypeptide constructs of the invention, and the singledomain antibody present in such polypeptide construct will also bereferred to herein as being in a “multiparatopic format”. Thus, forexample, a “biparatopic” polypeptide construct of the invention is apolypeptide construct that comprises at least one single domain antibodydirected against a first antigenic determinant on an antigen and atleast one further single domain antibody directed against a secondantigenic determinant on the same antigen, whereas a “triparatopic”polypeptide construct of the invention is a polypeptide construct thatcomprises at least one single domain antibody directed against a firstantigenic determinant on an antigen, at least one further Nanobodydirected against a second antigenic determinant on the same antigen andat least one further Nanobody directed against a third antigenicdeterminant on the same antigen; etc.

Accordingly, in its simplest form, a biparatopic polypeptide constructof the invention is a bivalent polypeptide construct of the invention(as defined herein), comprising a first single domain antibody directedagainst a first antigenic determinant on the antigen, and a secondsingle domain antibody directed against a second antigenic determinanton the same antigen, in which said first and second single domainantibody may optionally be linked via a linker sequence (as definedherein); whereas a triparatopic polypeptide of the invention in itssimplest form is a trivalent polypeptide of the invention (as definedherein), comprising a first single domain antibody directed against afirst antigenic determinant on the antigen, a second single domainantibody directed against a second antigenic determinant on the sameantigen and a third single domain antibody directed against a thirdantigenic determinant on the same antigen, in which said first, secondand third single domain antibody may optionally be linked via one ormore, and in particular one and more, in particular two, linkersequences.

Polypeptide constructs of the invention that contain at least two singledomain antibody, in which at least one single domain antibody isdirected against a first antigen and at least one single domain antibodyis directed against a second antigen (different from the first antigen),will also be referred to as “multispecific” polypeptides of theinvention, and the single domain antibody present in such polypeptideconstructs will also be referred to herein as being in a “multispecificformat”. Thus, for example, a “bispecific” polypeptide construct of theinvention is a polypeptide construct that comprises at least one singledomain antibody directed against a first antigen and at least onefurther single domain antibody directed against a second antigen (i.e.different from the first antigen), whereas a “trispecific” polypeptideof the invention is a polypeptide that comprises at least one singledomain antibody directed against a first antigen, at least one furthersingle domain antibody directed against a second antigen (i.e. differentfrom the first antigen) and at least one further single domain antibodydirected against a third antigen (i.e. different from both the first andthe second antigen); etc.

Accordingly, in its simplest form, a bispecific polypeptide of theinvention is a bivalent polypeptide of the invention (as definedherein), comprising a first single domain antibody directed against afirst antigen, and a second single domain antibody directed against asecond antigen, in which said first and second single domain antibodymay optionally be linked via a linker sequence (as defined herein);whereas a trispecific polypeptide of the invention in its simplest formis a trivalent polypeptide of the invention (as defined herein),comprising a first single domain antibody directed against a firstantigen, a second single domain antibody directed against a secondantigen and a third single domain antibody directed against a thirdantigen, in which said first, second and third single domain antibodymay optionally be linked via one or more, and in particular one andmore, in particular two, linker sequences.

For multivalent and multi specific polypeptides containing one or moreV_(HH) domains and their preparation, reference is also made to Conrathet al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans,Reviews in Molecular Biotechnology 74 (2001), 277-302; as well as to forexample WO 96/34103 and WO 99/23221. Some other examples of somespecific multispecific and/or multivalent polypeptide of the inventioncan be found in the applications by Ablynx N.V. referred to herein.

A “binding unit” as used herein refers to an amino acid sequence capableof binding an epitope. In the context of the present invention, abinding unit essentially consists of a single domain antibody (asdefined herein).

The term “single domain antibody” as used herein refers to a bindingsequence comprising an amino acid sequence that is suitable for use as adomain antibody and includes but is not limited to a “dAb” (or an aminoacid sequence that is suitable for use as a dAb) or a Nanobody® (asdescribed herein, and including but not limited to a V_(HH) sequence),other single variable domains such V_(H) or V_(L), or a suitablefragment of any one thereof.

The term “template polypeptide construct” as used herein refers to atheoretical or physically existing polypeptide construct, which is usedas a starting point for producing a diversity of polypeptide constructscomprising at least two single domain antibodies that are structuralvariants for the template polypeptide construct.

The term “identified single domain antibody” as used herein refers to asingle domain antibody, which as been generated previously (before themethod of the present invention is applied). In most cases, the“identified single domain antibody” has a specified amino acid sequenceand/or a specified antigen and/or epitope specificity.

The term “diversity” as used herein and particularly in the context of adiversity of polypeptide constructs refers to a set, group, collectionor library of polypeptide constructs which may contain any suitablenumber of sequences, such as 1, 2, 3 or about 5, 10, 50, 100, 500, 1000,5000, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ or more sequences. Depending on the one ormore desired characteristics of the polypeptide construct that one wouldlike to obtaine with the method of the invention, the skilled person canassess what the preferred number of sequences in the diversity ofpolypeptide construct. The preferred number of sequences in thediversity of polypeptide constructs may be about 5, about 10, about 50,about 100, or more than 1000, more than 10⁴, or more.

The term “antigen(s)” refers to the target molecule(s) recognized by theantigen-binding unit and more in particular by the antigen-binding siteof said antigen-binding unit.

The term “epitope(s)” refers to the particular site on the antigenrecognized by the antigen-binding unit (such as a binding unitessentially consisting of a (single) domain antibody or a polypeptideconstruct of the invention) and more in particular by theantigen-binding site of said antigen-binding unit, and can also bereferred to as the “antigenic determinant(s)”. Accordingly, the terms“epitope(s)” and “antigenic determinant(s)” may be used interchangeablyherein.

A binding unit, such as a single domain antibody or a polypeptideconstruct (or a fragment thereof), that can (specifically) bind to, thathas affinity for and/or that has specificity for a specific antigenicdeterminant, epitope, antigen or protein (or for at least one part,fragment or epitope thereof) is said to be “against” or “directedagainst” said antigenic determinant, epitope, antigen or protein.

In respect of a target or antigen, the term “interaction site” on thetarget or antigen means a site, epitope, antigenic determinant, part,domain or stretch of amino acid residues on the target or antigen thatis a site for binding to a ligand, receptor or other binding partner, acatalytic site, a cleavage site, a site for allosteric interaction, asite involved in multimerisation (such as homomerization orheterodimerization) of the target or antigen; or any other site,epitope, antigenic determinant, part, domain or stretch of amino acidresidues on the target or antigen that is involved in a biologicalaction or mechanism of the target or antigen. More generally, an“interaction site” can be any site, epitope, antigenic determinant,part, domain or stretch of amino acid residues on the target or antigento which a polypeptide construct of the invention can bind such that thetarget or antigen (and/or any pathway, interaction, signalling,biological mechanism or biological effect in which the target or antigenis involved) is modulated.

The term “specificity” refers to the number of different types ofantigens or antigenic determinants to which a particular antigen-bindingmolecule (such as a binding unit, e.g. a single domain antibody, or apolypeptide construct of the invention) can bind. The specificity of anantigen-binding molecule can be determined based on affinity and/oravidity.

All documents cited in the present specification are hereby incorporatedby reference in their entirety.

The Figures, Sequence Listing and the Experimental Part/Examples areonly given to further illustrate the invention and should not beinterpreted or construed as limiting the scope of the invention and/orof the appended claims in any way, unless explicitly indicated otherwiseherein.

In a first aspect, the invention provides methods for obtainingpolypeptide constructs directed against one or more antigens orepitopes. Such methods are based on the observation that single domainantibodies (as defined herein) can appropriately be used as buildingblocks in the generation of a diversity of polypeptide constructs, fromwhich suitable polypeptide constructs having one or more desiredcharacteristics can be obtained.

As will be clear from the further description herein, the inventioninvolves the use of binding units essentially consisting of singledomain antibodies as “building blocks” to form polypeptide constructs(such as, but without limitation, the bi- or multiparatopic, the bi- ormultivalent and/or the bi- or multispecific polypeptide constructs ofthe invention described herein), i.e. by suitably combining them withother groups, residues, moieties or binding units, such that the formedpolypeptide constructs of the present invention may exhibit one or moredesired characteristics or biological functions.

It is envisaged that in the polypeptide constructs of the invention,single domain antibodies can be linked to each other and/or optionallyto other groups, residues, moieties or binding units (e.g. viadisulphide bridges or via linker sequences, such as peptide linkersequences). Preferably, such one or more other groups, residues,moieties or binding units are amino acid sequences. As will become clearto the skilled person from the further disclosure herein, such furthergroups, residues, moieties may or may not provide further functionalityto the polypeptide constructs of the invention and may or may not modifythe properties of the polypeptide constructs of the invention. In thepolypeptide constructs of the invention, the one or more amino acidsequences of the invention and the one or more groups, residues,moieties or binding units may be linked directly to each other and/orvia one or more suitable linkers or spacers. For example, when the oneor more groups, residues, or moieties are amino acid sequences, thelinkers may also be amino acid sequences, so that the resultingpolypeptide construct is a fusion (protein) or fusion (polypeptide).

In particular, the invention provides methods for obtaining polypeptideconstructs having one or more, more particularly two or more desiredcharacteristics, wherein the polypeptide constructs comprise at leasttwo single domain antibodies and are directed against one or moreantigens or epitopes, which methods comprise at least the steps of: (i)selecting a template polypeptide construct, (ii) producing a diversityof polypeptide constructs that are structural variants for the selectedtemplate polypeptide construct, wherein said structural variants eachcomprise at least two single domain antibodies, and (iii) screening theproduced diversity of polypeptide constructs for polypeptide constructshaving the one or more, more particularly two or more desiredcharacteristics, wherein the polypeptide constructs comprise at leasttwo single domain antibodies and are directed against one or moreantigens and/or epitopes.

The step of selecting a template polypeptide construct may also involvedetermining a theoretical template, e.g. “a polypeptide constructcapable of binding to a certain antigen X of interest”. The selection ofa theoretical template implies that there are no particular structuralrequirements or preferences for the polypeptide construct with thedesired characteristics that one would like to obtain from the methodsof the invention and/or that no template polypeptide construct isavailable as a starting point for generating the diversity ofpolypeptide constructs.

Alternatively, the step of selecting a template antibody may involveselecting a physically existing (i.e. previously generated) polypeptideconstruct comprising at least one single domain antibody. In particularembodiments, the physically existing polypeptide construct is directedagainst the antigen or epitope of interest and the methods of theinvention comprise the generation of a diversity of polypeptideconstructs which comprises structural variants of the templatepolypeptide construct (as described herein below) from which apolypeptide construct can be selected according to the methods of theinvention having particular (additional) suitable and/or desiredcharacteristics, more particularly in addition to the feature of bindingto the antigen of interest. In alternative embodiments, the templatepolypeptide construct is a physically existing polypeptide constructdirected against one or more antigens or epitopes, which are differentfrom the antigen or epitopes of interest. In such embodiments selectioncan include selection based on a characteristic other than bindingaffinity e.g. solubility. Template polypeptide constructs combining oneor more physically existing or identified single domain antibodies withone or more theoretical single domain antibodies or linkers are alsoenvisaged to be selected in the methods of the invention.

Thus, in particular embodiments, methods for obtaining a polypeptideconstruct directed against antigen or epitope X may involve theselection of a particular template polypeptide construct comprising oneor more single domain antibodies directed against antigen or epitope X,the generation of a diversity of structural variants of polypeptideconstructs directed against antigen or epitope X (for the nature of thediversity see below), and the selection of a polypeptide constructdirected against antigen or epitope X, having one or more suitablecharacteristics, in addition to the binding to antigen or epitope X.

In further embodiments, methods of the invention are used for thegeneration of a polypeptide construct directed against more than oneantigen, for instance against antigens or epitopes X and Y. In suchmethods, the selection of the template polypeptide construct maycomprise the selection of a (physically existing or theoretical orcombined existing/theoretical) template polypeptide construct directed(ore envisaged to be directed) against the two or more antigens orepitopes of interest (X and Y), e.g. comprising two (identified) singledomain antibodies, each directed to one of the antigens or epitopes ofinterest (one single domain directed against X, one single domainantibody directed against Y). Such methods further comprise the step ofgenerating a diversity of structural variants of polypeptide constructsdirected against antigens or epitopes X and Y (this being determined bythe nature of the template polypeptide construct, see below), and theselection of a polypeptide construct directed against antigens orepitopes X and Y, having one or more suitable characteristics, inaddition to the binding to antigens or epitopes X and Y.

In these embodiments, the template polypeptide construct may be atheoretical construct or may comprise two or more single domainantibodies, possibly comprising one or more identified single domainantibodies known to be directed against X or Y. Accordingly, particularembodiments are envisaged where the template polypeptide constructcomprises at least one identified single domain antibody known to bedirected against one of the antigens (e.g. against antigen or epitope X)and at least one theoretical single domain antibody (e.g. a singledomain antibody envisaged to be directed against antigen or epitope Y),the step of generating a diversity of structural variants may comprisegenerating a diversity of combinations (i.e. fusion proteins) of (e.g.one or more copies of) the identified single domain antibody known to bedirected against X with e.g. a collection of different single domainantibodies (optionally directed against the other antigen or epitope Y,or random sequences which can be screened for binding to antigen orepitope Y). Alternatively, the template polypeptide construct maycomprise two or more identified (i.e. previously generated) singledomain antibodies each against one of both antigens or epitopes and thestep of generating a diversity of structural variants may comprisegenerating polypeptide constructs comprising different combinations ofthe identified single domain antibodies (e.g. differing in number and/orrelative position in the construct), and/or constructs comprisingdifferent modifications of (such as modifications of the CDR or FRregions of) either one or both of the identified single domainantibodies. In the selection step, a polypeptide construct having, inaddition to the ability to recognize X and Y, one or more suitablecharacteristics such as suitable affinity (against X and/or Y),solubility etc. (as further described herein).

Accordingly, it is envisaged that the template polypeptide constructneed not determine the number or the nature of the single domainantibodies in the diversity of polypeptide constructs generated nor inthe polypeptide construct one would like to obtain from the methods ofthe invention. More particularly, it is noted that for the purpose ofgenerating a polypeptide construct with improved affinity against anantigen, a template antibody can be selected which comprises one(physically existing or theoretical) single domain antibody against theantigen. In such embodiments, the step of producing a diversity ofpolypeptide constructs may comprise generating polypeptide constructscomprising different numbers of the same single domain antibody and/ordifferent combinations of single domain antibodies directed againstdifferent epitopes of the same antigen, with the aim of obtaining apolypeptide construct with the desired suitable affinity for theantigen. The same applies when the methods of the invention are used forobtaining polypeptide constructs directed against two or more antigens.

Methods according to the present invention further comprise step (ii) ofproducing a diversity of polypeptide constructs that are structuralvariants of the selected template polypeptide construct. A diversity ofstructural variants of the template polypeptide construct according tothe invention comprises different polypeptide constructs, eachcomprising at least two single domain antibodies that may be linked toeach other and optionally to one or more other groups, residues,moieties or binding units.

Depending on the nature of the characteristic(s) desired for thepolypeptide construct that one envisages to obtain with methodsaccording to the invention, the produced diversity can comprisedifferent types of structural variants for the template polypeptideconstruct. As detailed above, where the template polypeptide constructcomprises one or more identified (i.e. previously generated) singledomain antibodies, linkers or other structures or moieties, thediversity may comprise structural variants of the template polypeptideconstruct. Where the template polypeptide construct is a theoreticalconstruct, the diversity needs to be generated starting from differentnewly generated single domain antibodies, linkers, structures ormoieties.

In particular embodiments, the step of producing a diversity ofstructural variants of said template polypeptide construct may compriseproducing a diversity of structural variants with regard to the numberand/or identity of single domain antibodies in the polypeptideconstructs. In particular embodiments, the template polypeptideconstruct comprises one single domain antibody and producing a diversityof structural variants for the template polypeptide construct mayinvolve producing a diversity of antibody cosntructs comprising at leasttwo single domain antibodies but more particularly three, four, five,six or more single domain antibodies.

According to particular embodiments the template antibody comprises anidentified (i.e. previously generated) single domain antibody, and theone or more of the additional single domain antibodies present in thediversity of polypeptide constructs are either the same as or differentfrom the identified single domain antibody envisaged in the templateconstruct and may be the same or may be different from each other. Forinstance, where the template polypeptide construct is selected tocomprise one single domain antibody of identity A, a diversity ofstructural variants with regard to the number of single domainantibodies may comprise polypeptide constructs comprising, in additionto single domain antibody A, one or more additional single domainantibodies of types A. Such structural variants may further be withregard to both the number and the identity of the single domain antibodyA, such that the diversity of structural variants may comprisepolypeptide constructs comprising, in addition to single domain antibodyA, one or more single domain antibodies B, C, D, etc (such as e.g.without being limiting A-A, A-A-A, A-A-A-A, A-B, A-A-B, A-A-A-B, A-B-C,A-A-B-C, A-B-C-D, etc.). A similar diversity may be generated startingfrom a template polypeptide construct comprising two or more singledomain antibodies.

Accordingly, in particular embodiments, the production of a diversity ofstructural variants for the template polypeptide construct may compriseobtaining structural variants having fewer, an equal number of or moresingle domain antibodies compared to the template polypeptide construct,wherein the single domain antibodies may be the same or may be differentfrom each other, provided that each structural variant of the diversitycomprises at least two single domain antibodies.

In further particular embodiments, the methods of the inventionencompass generating a diversity of structural variants with regard tothe relative position of single domain antibodies within the polypeptideconstructs. According to more particular embodiments, a templatepolypeptide construct is selected comprising one or more single domainantibodies having a certain relative position within the templatepolypeptide construct, and the generation of a diversity of structuralvariants comprises generating polypeptide constructs comprising the samesingle domain antibodies of the template polypeptide construct, but withdifferent relative positions within these structural variants comparedto the template polypeptide construct. For instance, when a particulartemplate polypeptide construct is selected comprising three singledomain antibodies A, B and C that are positioned relative to one anotherin a configuration A-B-C (wherein “-” represents the linkage, e.g. via alinker sequence such as a peptide linker, between A and B), a diversityof structural variants may comprise structural variants comprising thesame three single domain antibodies A, B and C positioned relative toone another in one of the configurations A-B-C, B-A-C, or B-C-A, orA-C-B, or C-A-B, or C-B-A.

As indicated above, it is further envisaged that structural variants mayinvolve variants with regard to additional groups which can influencethe desired characteristics of the polypeptide construct. Accordingly,in particular embodiments, the diversity of polypeptide constructscomprises variants with regard to the relative position of the singledomain antibodies, including variants with respect to other groups,residues, moieties present in the polypeptide constructs. For instance,when a particular template polypeptide construct is selected comprisingtwo single domain antibodies A and B that are positioned relative to oneanother in a configuration A-B (wherein “-” represents the linkage, e.g.via a linker sequence such as a peptide linker, between A and B), adiversity of structural variants may comprise structural variantscomprising the same two single domain antibodies A and B positionedrelative to one another in one of the configurations A-B, B-A, or . . .X-A-B, X-B-A, or A-B-X, B-A-X . . . or A-X-B, B-X-A, or B-X-X- . . . -A,or A-X-X- . . . B, wherein X may be other groups, moieties present inthe diversity of structural variants.

In yet further particular embodiment, the generation of a diversity ofstructural variants for template polypeptide construct comprisesgenerating a diversity of structural variants with regard to the aminoacid residues of the CDR regions of the single domain antibodies in thepolypeptide constructs. For instance, where a template antibody isselected comprising one or more single domain antibodies that arecharacterized by three specific CDR regions, a diversity of structuralvariants for the template polypeptide construct may be generated byproviding different polypeptide constructs comprising the one or moresingle domain antibodies of the template polypeptide construct wherebyin at least one of the three CDR regions of at least one of the singledomain antibodies, one or more amino acid residues are differentcompared to the corresponding amino acid residues in the correspondingCDR regions in the template polypeptide construct.

For instance, starting from a template polypeptide construct comprisingtwo or more identified (i.e. previously generated) single domainantibodies, each comprising three CDR regions, a diversity of structuralvariants with regard to the amino acid residues of the CDR regions ofthe single domains antibodies in the polypeptide construct may comprisea diversity of structural variants all comprising essentially the samesingle domain antibodies but having in at least one of the three CDRregions (such as in one, e.g. preferably in CDR3, in two, e.g.preferably in CDR2 and CDR3, or in all three) of at least one of thesingle domain antibodies, at least one amino acid substitution (i.e. anamino acid residue which has been replaced by another amino acidresidue).

In a further particular embodiment, where the selected templatepolypeptide construct comprises two or more identified (i.e. previouslygenerated) single domain antibodies, each comprising three CDR regions,a diversity of structural variants of the template polypeptide constructwith regard to the amino acid residues of the CDR regions of the singledomain antibodies in the polypeptide construct may comprise differentpolypeptide constructs each comprising essentially the same singledomain antibodies, but having in at least one (such as in one, e.g.preferably in CDR3, in two, e.g. preferably in CDR2 and CDR3, or in allthree) of the three CDR regions of at least one of the single domainantibodies, at least one amino acid residue which is deleted or added,compared to the template polypeptide construct.

For example, one or more of the CDR regions in the single domainantibody may be altered in order to provide single domain antibodiesand/or polypeptide construct with increased affinity compared to thewild type single domain antibody and/or polypeptide construct (alsoreferred to as affinity maturation). Alterations of the CDR regions mayinclude (without being limiting) the addition, deletion and/or changingof one or more of the amino acid residues in the CDR region (e.g.applying point mutations at certain specified positions); CDR grafting,veneering, the (partially or fully) randomization of the amino acidresidues in the CDR); DNA shuffling, chain shuffling, look-throughmutagenesis, walk-through mutagenesis and any other technique known inthe art or any suitable combination of any of the foregoing. Referenceis for example made to (without being limiting) the techniques describedin WO 91/15581, WO 05/003345, International applications by Ablynx N.V.PCT/EP2008/058617 and PCT/EP2008/058618 as well as U.S. provisionalapplication 61/077,924 by Albynx N.V. filed on 7 Jul. 2008 entitled“Methods for providing improved immunoglobulin sequences”.

Such diversity of polypeptide constructs comprising an alteration in oneor more of the CDR regions can be generated by any method known in theart (such as e.g. PCR assembly of an appropriate series or pool ofolignonucleotides and similar techniques for engineering immunoglobulinsequences well known to the skilled person, followed by suitableexpression).

In yet further particular embodiments of the methods described herein,the step of producing a diversity of polypeptide constructs startingfrom a template polypeptide construct comprising e.g. two or moreidentified (i.e. previously generated) single domain antibodiescomprises producing a diversity of structural variants of thepolypeptide construct with regard to the amino acid residues of theframework regions of the single domain antibodies in the polypeptideconstructs. For instance, a diversity of structural variants of atemplate polypeptide construct comprising two or more single domainantibodies that are each characterized by four specific frameworkregions, may be a diversity of structural variants wherein, for one orboth of the single domain antibodies, in at least one of the fourframework regions one or more amino acid residues are different comparedto the corresponding amino acid residues in the corresponding frameworkregions of the template polypeptide construct.

For instance, starting from a template polypeptide construct comprisingtwo or more identified (i.e. previously generated) single domainantibodies, each comprising four framework regions, producing adiversity of structural variants with regard to the amino acid residuesof the framework regions of the single domain antibodies, may compriseproducing a diversity of polypeptide constructs comprising theessentially the same single domain antibodies but wherein in at leastone (in one (such as in FR2 or FR4), in two (such as in FR2 and FR4 orin FR2 and FR3 or in FR3 and FR4), in three (such as in FR2, FR3 andFR4) or in all four) of the four framework regions (of one or more, suchas two, three or more of the single domain antibodies) at least oneamino acid residue has been replaced by another amino acid residue.

In further envisaged embodiments, when a template polypeptide constructis selected comprising two or more single domain antibodies, eachcomprising four framework regions, a diversity of structural variants ofsaid template polypeptide construct with regard to the amino acidresidues of the framework regions of the single domain antibodies in thepolypeptide constructs, may comprise a diversity of structural variantscomprising the same single domain antibodies, wherein in at least one(in one (such as in FR2 or FR4), in two (such as in FR2 and FR4 or inFR2 and FR3 or in FR3 and FR4), in three (such as in FR2, FR3 and FR4)or in all four) of the four framework regions (in one or more, such asone, two, three or more of the single domain antibodies of theconstruct) at least one amino acid residue has been added or deleted.

In a specific embodiment the amino acid sequence of the frameworkregions may be altered by “camelization” of specific amino acid residuesin the framework regions. Camelization refers to the replacing orsubstitution of one or more amino acid residues in the amino acidsequence of a (naturally occurring) V_(H) domain from a conventional4-chain antibody by one or more of the amino acid residues that occur atthe corresponding position(s) in a V_(HH) domain of a heavy chainantibody. This can be performed in a manner known per se, which will beclear to the skilled person, for example on the basis of the furtherdescription herein. Such “camelizing” substitutions are preferablyinserted at amino acid positions that form and/or are present at theV_(H)-V_(L) interface, and/or at the so-called Camelidae hallmarkresidues, as defined herein (see for example WO 94/04678, Davies andRiechmann FEBS Letters 339: 285-290, 1994; Davies and Riechmann ProteinEngineering 9 (6): 531-537, 1996; Riechmann J. Mol. Biol. 259: 957-969,1996; and Riechmann and Muyldermans J. Immunol. Meth. 231: 25-38, 1999).

In a specific embodiment the amino acid sequence of the frameworkregions may be altered by “humanization” of specific amino acid residuesin the framework regions. In particular, humanized single domainantibodies may be single domain antibodies in which at least one aminoacid residue is present (and in particular, in at least one of theframework residues) that is and/or that corresponds to a humanizingsubstitution. Potentially useful humanizing substitutions can beascertained by comparing the sequence of the framework regions of anaturally occurring single domain antibody sequence with thecorresponding framework sequence of one or more closely related humanV_(H) sequences. The potentially useful humanizing substitutions (orcombinations thereof) thus determined can be introduced into thepolypeptide construct comprising said single domain antibody sequence(in any manner known per se, as further described herein). The resultingdiversity of polypeptide constructs comprising at least one humanizingsubstitution in at least one single domain antibody can subsequently bescreened for one or more desired properties.

In yet further particular embodiments of the methods of the invention, atemplate polypeptide construct is selected comprising two or more singledomain antibodies, and the generation of a diversity of structuralvariants of the template polypeptide construct may also involveproducing a diversity at the DNA level, i.e. a diversity of structuralvariants with regard to the codon usage in the selected templatepolypeptide construct. For instance, if a template polypeptide constructis characterized by an amino acid sequence that is obtained byexpressing a particular nucleic acid sequence, wherein the nucleic acidsequence consists of a sequential number of codons, a diversity ofstructural variants may comprise a diversity of nucleic acid sequencesencoding the same amino acid sequences wherein in each of the nucleicacid sequences at least one of the codons is different compared to thecorresponding codons in the nucleic acid sequence coding for the aminoacid sequence of the template polypeptide construct.

For instance, where a template polypeptide construct is selectedcomprising one or more single domain antibody amino acid sequences whichare encoded by one or more selected nucleic acid sequences, the nucleicacid sequences consisting of a sequential number of codons, the step ofproducing a diversity of structural variants may involve producingstructural variants wherein one or more nucleotide base pair changeshave been introduced, such as by substitution, deletion or addition ofone or more base pairs, compared to the one or more sequences encodingthe template polypeptide construct. As a consequence, producing adiversity of structural variants of a template construct antibody withregard to codon usage may imply introducing variations at the nucleotidesequence level of the template polypeptide construct and accordingly mayor may not result in variations in the amino acid sequences encoded bythe structural variants.

According to further embodiments of the methods of the presentinvention, the step of producing a diversity of structural variants maycomprise producing a diversity of structural variants and/or variationswhich involve more than one type of variants and/or variations describedabove, i.e. a combination of structural variants or variations such asbut not limited to the following combinations of variants and/orvariations, which include combinations of:

-   -   the number of the single domain antibodies in the polypeptide        constructs (as described above) and the relative position of        said single domain antibodies within said polypeptide constructs        (as described above); or    -   the number and/or identity of the single domain antibodies in        said polypeptide constructs (as described above) and the amino        acid residues of the CDR regions of one or more single domain        antibodies in the polypeptide constructs (as described above);        or    -   the number of the single domain antibodies in the polypeptide        constructs (as described above) and the amino acid residues of        the framework region(s) of one or more of the single domain        antibodies in the polypeptide constructs (as described above);        or    -   the number and/or identity of the single domain antibodies in        the polypeptide constructs (as described above) and the codon        usage in the sequence encoding the selected template polypeptide        construct (as described above); or    -   the relative position of the single domain antibodies within the        polypeptide constructs (as described above) and the amino acid        residues of the CDR region(s) of one or more of the single        domain antibodies in the polypeptide constructs (as described        above); or    -   the relative position of the single domain antibodies within the        polypeptide constructs (as described above) and the amino acid        residues of the framework region(s) of one or more of the single        domain antibodies in the polypeptide constructs (as described        above); or    -   the relative position of the single domain antibodies within the        polypeptide constructs (as described above) and the codon usage        in the nucleic acid sequences encoding the polypeptide        constructs; or    -   the amino acid residues of the CDR region(s) of one or more of        the single domain antibodies in the polypeptide constructs (as        described above) and the amino acid residues of the framework        region(s) of one or more of the single domain antibodies in the        polypeptide constructs (as described above); or    -   the amino acid residues of the CDR region(s) of one or more of        the single domain antibodies in the polypeptide constructs (as        described above) and the codon usage in the nucleic acid        sequences encoding the polypeptide constructs; or    -   the amino acid residues of the framework region(s) of one or        more of the single domain antibodies in the polypeptide        constructs (as described above) and the codon usage in the        nucleic acid sequences encoding the polypeptide constructs; or    -   the number of said single domain antibodies in the polypeptide        constructs (as described above), the relative position of the        single domain antibodies within said polypeptide constructs (as        described above) and the amino acid residues of the CDR        region(s) of one or more of the single domain antibodies in the        polypeptide constructs (as described above); or    -   the number and/or identity of the single domain antibodies in        the polypeptide constructs (as described above), the relative        position of the single domain antibodies within said polypeptide        constructs (as described above) and the amino acid residues of        the framework region(s) of the single domain antibodies in the        polypeptide constructs (as described above); or    -   the number and/or identity of the single domain antibodies in        the polypeptide constructs (as described above), the relative        position of the single domain antibodies within the polypeptide        constructs (as described above) and the codon usage in the        nucleic acid sequences encoding the polypeptide constructs; or    -   the number and/or identity of the single domain antibodies in        the polypeptide constructs (as described above), the amino acid        residues of the CDR region(s) of on or more of the single domain        antibodies in the polypeptide constructs (as described above)        and the amino acid residues of the framework region(s) of one or        more of the single domain antibodies in the polypeptide        constructs (as described above); or    -   the number and/or identity of the single domain antibodies in        the polypeptide constructs (as described above), the amino acid        residues of the CDR region(s) of one or more of the single        domain antibodies in the polypeptide constructs (as described        above) and the codon usage in the nucleic acid sequences        encoding the polypeptide constructs; or    -   the number and/or identity of the single domain antibodies in        the polypeptide constructs (as described above), the amino acid        residues of the framework region(s) of one or more of the single        domain antibodies in the polypeptide constructs (as described        above) and the codon usage in the nucleic acid sequences        encoding the polypeptide constructs; or    -   the relative position of the single domain antibodies within the        polypeptide constructs (as described above), the amino acid        residues of the CDR region(s) of one or more of the single        domain antibodies in the polypeptide constructs (as described        above) and the amino acid residues of the framework region(s) of        one or more of the single domain antibodies in the polypeptide        constructs (as described above); or    -   the relative position of one or more of the single domain        antibodies within the polypeptide constructs (as described        above), the amino acid residues of the CDR region(s) of one or        more of the single domain antibodies in said polypeptide        constructs (as described above) and the codon usage in the        nucleic acid sequences encoding the polypeptide constructs; or    -   the relative position of the single domain antibodies within the        polypeptide constructs (as described above), the amino acid        residues of the framework region(s) of one or more of the single        domain antibodies in the polypeptide constructs (as described        above) and the codon usage in the nucleic acid sequences        encoding the polypeptide constructs; or    -   the amino acid residues of the CDR region(s) of one or more of        the single domain antibodies in the polypeptide constructs (as        described above), the amino acid residues of the framework        region(s) of one or more of the single domain antibodies in the        polypeptide constructs (as described above) and the codon usage        in the nucleic acid sequences encoding the polypeptide        constructs; or    -   the number and/or identity of the single domain antibodies in        the polypeptide constructs (as described above), the relative        position of the single domain antibodies within the polypeptide        constructs (as described above), the amino acid residues of the        CDR region(s) of one or more of the single domain antibodies in        the polypeptide constructs (as described above) and the amino        acid residues of the framework region(s) of one or more of the        single domain antibodies in the polypeptide constructs (as        described above) or;    -   the number and/or identity of the single domain antibodies in        the polypeptide constructs (as described above), the relative        position of the single domain antibodies within the polypeptide        constructs (as described above), the amino acid residues of the        CDR region(s) of one or more of the single domain antibodies in        the polypeptide constructs (as described above) and the codon        usage in the nucleic acid sequences encoding the polypeptide        constructs; or    -   the number and/or identity of the single domain antibodies in        the polypeptide constructs (as described above), the relative        position of the single domain antibodies within the polypeptide        constructs (as described above), the amino acid residues of the        framework region(s) of one or more of the single domain        antibodies in the polypeptide constructs (as described above)        and the codon usage in the nucleic acid sequences encoding the        polypeptide constructs; or    -   the amino acid residues of the CDR region(s) of one or more of        the single domain antibodies in the polypeptide constructs (as        described above), the amino acid residues of the framework        region(s) of one or more of the single domain antibodies in the        polypeptide constructs (as described above), the codon usage in        the nucleic acid sequences encoding the polypeptide constructs        and the number and/or identity of the single domain antibodies        in the polypeptide constructs (as described above); or    -   the amino acid residues of the CDR region(s) of one or more of        the single domain antibodies in the polypeptide constructs (as        described above), the amino acid residues of the framework        region(s) of one or more of the single domain antibodies in the        polypeptide constructs (as described above), the codon usage in        the nucleic acid sequences encoding the polypeptide constructs        and the relative position of the single domain antibodies within        the polypeptide constructs (as described above) or;    -   the amino acid residues of the CDR region(s) of one or more of        the single domain antibodies in the polypeptide constructs (as        described above), the amino acid residues of the framework        region(s) of one or more of the single domain antibodies in the        polypeptide constructs (as described above), the codon usage in        the nucleic acid sequences encoding the polypeptide constructs,        the relative position of the single domain antibodies within the        polypeptide constructs (as described above) and the number        and/or identity of the single domain antibodies in the        polypeptide constructs (as described above).

The length, the degree of flexibility and/or other properties of thelinker(s) used in polypeptide constructs may have some influence on theproperties of the final polypeptide construct of the invention,including but not limited to the affinity, specificity or avidity forone or more particular antigens or epitopes.

Therefore, the methods of the present invention, also encompass in step(ii) producing a diversity of polypeptide constructs that are structuralvariants for the selected template polypeptide construct of step (i)comprises producing structural variants with regard to the peptidelinkers. In particular embodiments modifying the peptide linkers mayfavorably affect one or more desirable characteristics of thepolypeptide construct.

Accordingly, in further particular embodiments of step (ii) the methodsdescribed herein comprising producing a diversity of polypeptideconstructs that are structural variants for the selected templatepolypeptide construct, comprises producing structural variants accordingto one or more of the following (non limiting) examples:

-   -   structural variants with regard to the composition and/or length        of the one or more peptide linkers, and/or,    -   structural variants with regard to the number of the one or more        linkers, and/or,    -   structural variants with regard to the relative position of the        one or more linkers in the polypeptide constructs.

As described above, the template polypeptide construct selected in themethods of the present invention may be a theoretical templateconstruct, comprising one or more single domain antibodies that areenvisaged to be linked to each other and/or to other groups or moietiesvia suitable (peptide) linkers or spacers. Alternatively, the templatepolypeptide construct may be an isolated polypeptide construct,comprising one or more identified (i.e. previously generated) singledomain antibodies that are linked to each other and/or to other groupsor moieties via actual physical linkers and/or spacers.

Suitable spacers or linkers for use in multivalent (and optionallymultispecific or multiparatopic) polypeptide constructs will be clear tothe skilled person, and may generally be any linker or spacer used inthe art to link amino acid sequences. Preferably, the linker and/orspacer is suitable for use in producing polypeptide constructs that areintended for pharmaceutical use.

Some particularly suitable linkers or spacers include the linkers and/orspacers that are used in the art to link antibody fragments or antibodydomains. These include the linkers generally known in the art, as wellas for example linkers that are used in the art to construct diabodiesor ScFv fragments (in this respect, however, its should be noted that,whereas in diabodies and in ScFv fragments, the linker sequence usedshould have a length, a degree of flexibility and other properties thatallow the pertinent V_(H) and V_(L) domains to come together to form thecomplete antigen-binding site, there is no particular limitation on thelength or the flexibility of the linker used in the polypeptideconstructs of the invention, since each single domain antibody by itselfforms a complete antigen-binding site).

Some preferred examples of such amino acid sequences include gly-serlinkers, for example of the type (gly_(x)ser_(y))_(z), such as (forexample (gly₄ser)₃ or (gly₃ser₂)₃, as described in WO 99/42077 and theGS30, GS15, GS9 and GS7 linkers described in the applications by Ablynxmentioned herein (see for example WO 06/040153 and WO 06/122825), aswell as hinge-like regions, such as the hinge regions of naturallyoccurring heavy chain antibodies or similar sequences (such as describedin WO 94/04678).

Some other particularly preferred linkers are poly-alanine (such asAAA), as well as the linkers GS35, GS30 (SEQ ID NO: 85 in WO 06/122825)and GS9 (SEQ ID NO: 84 in WO 06/122825).

Other suitable linkers generally comprise organic compounds or polymers,in particular those suitable for use in proteins for pharmaceutical use.For instance, polyethyleneglycol) moieties have been used to linkantibody domains, see for example WO 04/081026.

According to particular embodiments of the methods of the presentinvention, the step of producing a diversity of polypeptide constructsthat are structural variants for the selected template polypeptideconstruct comprising at least two single domain antibodies that arelinked via one or more peptide linkers, comprises producing structuralvariants with regard to the composition and/or length of the one or morepeptide linkers. In this respect, the different linkers in the diversityof polypeptide constructs may have one or more different amino acidresidues in their amino acid sequence or they may have less or moreamino acid residues resulting in a different linker length compared tothe template polypeptide construct.

For instance, the step of producing a diversity of polypeptideconstructs may comprise producing structural variants with regard to theamino acid sequence of the one or more peptide linkers in the templatepolypeptide construct.

Accordingly, starting from a template polypeptide construct comprisingtwo or more identified (i.e. previously generated) single domainantibodies linked via one or more peptide linkers, generating adiversity of structural variants with regard to the amino acid sequenceof the one or more peptide linkers, may comprise generating a diversityof polypeptide constructs comprising essentially the same linkers butwherein in at least one of the linkers at least one amino acid residuehas been replaced by another amino acid residue.

For instance, producing a diversity of structural variants ofpolypeptide constructs with regard to the amino acid sequence of the oneor more peptide linkers may involve producing structural variantscomprising different types of Gly-Ser linkers, for example differenttypes of (Gly_(x)Ser_(y))_(z).

Alternatively, the step of producing a diversity of polypeptideconstructs that are structural variants for the selected templatepolypeptide construct comprising at least two single domain antibodiesthat are linked via one or more peptide linkers may comprise producingstructural variants with regard to the length of the one or more peptidelinkers.

Accordingly, starting from a template polypeptide construct comprisingtwo or more identified (i.e. previously generated) single domainantibodies linked via one or more peptide linkers, generating adiversity of structural variants with regard to the length of said oneor more peptide linkers, may comprise generating a diversity ofpolypeptide constructs comprising essentially the same linkers butwherein in at least one of the linkers at least one amino acid residuehas been added and/or deleted.

For instance, producing a diversity of structural variants ofpolypeptide constructs with regard to the length of the one or morepeptide linkers may involve producing structural variants comprisingpeptide linkers having a length varying between 1 and 50, such as alength varying between 1 and 35, or such as a length varying between 1and 10 amino acid residues (such as e.g. Gly-Ser type linkers(Gly₄Ser)₃, (Gly₄S)₅, (Gly₄Ser)₆, etc.).

It is also encompassed in particular embodiments of the methods of thepresent invention that the step of producing a diversity of polypeptideconstructs that are structural variants with regard to the compositionand/or length of the one or more peptide linkers in the polypeptideconstructs, may comprise producing structural variants with regard tothe amino acid sequence of the one or more peptide linkers as well aswith regard to the length of the one or more peptide linkers (such ase.g. Gly-Ser type linkers (Gly₄Ser)₃, (Gly₄S)₅, (Gly₄Ser)₆, etc. and(Gly₄Ser)₃ or (Gly₃Ser₂)₃).

Thus, where the template polypeptide construct comprises two or moreidentified (i.e. previously produced) single domain antibodies linkedvia one or more peptide linkers, generating a diversity of structuralvariants with regard to the amino acid sequence composition as well aswith regard to the length of said one or more peptide linkers, maycomprise generating a diversity of polypeptide constructs comprisingessentially the same linkers but wherein in at least one of the linkersat least one amino acid residue has been substituted, added and/ordeleted.

According to further particular embodiments of the methods of thepresent invention, the step of producing a diversity of polypeptideconstructs that are structural variants for the selected templatepolypeptide construct comprising at least two single domain antibodiesthat are linked via one or more peptide linkers, comprises producingstructural variants with regard to the number of said one or morepeptide linkers.

For instance, starting from a template polypeptide construct comprisingtwo identified (i.e. previously produced) single domain antibodieslinked via one peptide linker, generating a diversity of structuralvariants with regard to the number of said one or more peptide linkers,may comprise generating a diversity of polypeptide constructs comprisingthe same two single domain antibodies that are directly linked, i.e.without using said one peptide linker.

Also, for example starting from a template polypeptide constructcomprising three identified (i.e. previously generated) single domainantibodies linked via two peptide linkers, generating a diversity ofstructural variants with regard to the number of said one or morepeptide linkers, may comprise generating a diversity of polypeptideconstructs comprising the same three single domain antibodies, whereinsaid diversity of structural variants comprises none or one peptidelinker.

Alternatively, starting from a template polypeptide construct comprisingtwo identified (i.e. previously generated) single domain antibodies oneor more other groups, moieties or binding units, wherein saidpolypeptide construct comprises three peptide linkers, generating adiversity of structural variants with regard to the number of said oneor more peptide linkers, may comprise generating a diversity ofpolypeptide constructs comprising the same two single domain antibodiesone or more other groups, moieties or binding units, wherein saiddiversity of structural variants comprises variants without linker,and/or variants with one or more peptide linkers, such as none, one,two, three, four, five, six, etc., wherein the one or more peptidelinkers may link (a) said single domain antibodies to each other, (b)said one or more groups, moieties or binding units to each other, or (c)may interlink said single domain antibodies to said one or more othergroups, moieties or binding units.

It should be stressed that the present embodiment also encompasses theproduction of a diversity of polypeptide constructs wherein one or morelinkers are present or absent and thus a diversity of polypeptideconstructs comprising polypeptide constructs wherein the single domainantibodies are directly linked to each other (or to one or more othergroups, moieties or binding units) as well as comprising polypeptideconstructs wherein the single domain antibodies are linked via a(peptide) linker to each other (or to one or more other groups, moietiesor binding units).

According to yet further particular embodiments of the methods of thepresent invention, the step of producing a diversity of polypeptideconstructs that are structural variants for the selected templatepolypeptide construct comprising at least two single domain antibodiesthat are linked via one or more peptide linkers, comprises producingstructural variants with regard to the relative position of said one ormore peptide linkers in the polypeptide constructs.

Accordingly, a template polypeptide construct may be selected comprisingone or more peptide linkers having a certain relative position withinthe template polypeptide construct, and the generation of a diversity ofstructural variants comprises generating polypeptide constructscomprising the same peptide linkers as present in the templatepolypeptide construct, but wherein the peptide linkers have differentrelative positions within these structural variants compared to thetemplate polypeptide construct.

For instance, when a particular template polypeptide construct isselected comprising four identified single domain antibodies (e.g. A, B,C, D) that are linked via three peptide linkers (e.g. “-”, “---” and“-------”) that are positioned relative to one another in a particularconfiguration (e.g. A-B---C-------D), a diversity of structural variantsmay comprise structural variants comprising the same four single domainantibodies (e.g. A, B, C and D) that are linked via the same threepeptide linkers (e.g. “-”, “---” and “-------”), wherein said peptidelinkers are positioned relative to one another in one of the followingnon-limiting configurations A-B---C------D, A---B-C-------D,A-B-------C---D, A---B-------C-D, A-------B-C---D, or A-------B---C-D.

As indicated above, it is further envisaged that structural variants mayinvolve variants with regard to additional groups which can influencethe desired characteristics of the polypeptide construct. Accordingly,in particular embodiments, the diversity of polypeptide constructscomprises variants with regard to the relative position of the one ormore peptide linkers, including with respect to one or more othergroups, moieties or binding units present in the polypeptide constructs.For instance, when a particular template polypeptide construct isselected comprising two identified single domain antibodies (e.g. A andB) and one other group, moiety or binding unit (e.g. X), linked to eachother via two peptide linkers (e.g. “---” and “----------”) that arepositioned relative to one another in a particular configuration (e.g.A----B----------X and A----B----------X), a diversity of structuralvariants may comprise structural variants comprising the same two singledomain antibodies (e.g. A and B) and the same one other group or moiety(e.g. X), linked to each other via the same two peptide linkers (e.g.“----” and “----------”), wherein said peptide linkers are positionedrelative to one another in the configuration (e.g. A----------B-----X).

According to further embodiments of the methods of the presentinvention, the step of producing a diversity of structural variants maycomprise generating a diversity of structural variants which involvemore than one type of variation of the one or more peptide linkers, i.e.a combination of structural variations such as but not limited to thefollowing combinations of variations, which include combinations of:

-   -   the composition and/or length of the one or more peptide linkers        in the polypeptide constructs (as described above) and the        number of the one or more peptide linkers in the polypeptide        constructs (as described above); or    -   the composition and/or length of the one or more peptide linkers        in the polypeptide constructs (as described above) and the        relative position of the one or more peptide linkers in the        polypeptide constructs (as described above); or    -   the number of the one or more peptide linkers in the polypeptide        constructs (as described above) and the relative position of the        one or more peptide linkers in the polypeptide constructs (as        described above); or    -   the composition and/or length of the one or more peptide linkers        in the polypeptide constructs (as described above), the number        of the one or more peptide linkers in the polypeptide constructs        (as described above) and the relative position of the one or        more peptide linkers in the polypeptide constructs (as described        above).

It will be understood to the skilled person that methods are equallyenvisaged wherein, upon generating a diversity of polypeptideconstructs, said diversity of polypeptide constructs may encompass bothstructural variants with regard to both the single domain antibodies aswell as structural variants with regard to the (peptide) linkers orspacers.

For example, in multivalent polypeptide constructs of the invention thatcomprise single domain antibodies directed against a multimeric antigen(such as a multimeric receptor, ligand or other protein), the length andflexibility of the linker are preferably such that it allows each singledomain antibody of the invention present in the polypeptide construct tobind to the antigenic determinant on each of the subunits of themultimer. Similarly, in a multispecific polypeptide construct of theinvention that comprises single domain antibodies directed against twoor more different antigenic determinants on the same antigen (forexample against different epitopes of an antigen and/or againstdifferent subunits of a multimeric receptor, channel or protein), thelength and flexibility of the linker are preferably such that it allowseach single domain antibody to bind to its intended antigenicdeterminant. Based on the disclosure herein, the skilled person will beable to determine the optimal linker(s) for use in a specificpolypeptide of the invention.

This particular embodiment of the present invention is particularlysuited for the selection of or screening for polypeptide constructcomprising at least two single domain antibodies that preferentiallyshow intramolecular binding to a certain antigen compared tointermolecular binding. By “intramolecular” binding is meant that thepolypeptide construct of the invention can simultaneously bind twoepitopes on the same antigen (these two epitope can be the same, e.g. ifthe antigen is a multimer; or these epitopes may be different, e.g. whenthe method of the invention is used for screening multipartopicpolypeptide constructs (as is further defined herein)).

The choice of linker length in biparatopic, triparatopic ormultiparatopic polypeptides of the invention can also be such that onlya limited epitope space on the antigen is covered. Linker lengthrestriction can, for example, help to avoid targeting epitopes whichshould not be neutralized (e.g. those essential for a function of theantigen) or to target regions relatively adjacent to a first ‘guiding’single domain antibody.

The choice of the format (N- or C-terminal position of the differentsingle domain antibodies) of the biparatopic, triparatopic ormultiparatopic polypeptides of the invention and linker length can alsobe used to obtain molecules that bind avidly to the target antigen (viatwo, or more, binding sites), yet are purposely not agonistic. Byoptimising the format and linker length and composition, the bindingsites can be positioned in such way that simultaneous binding of two ormore single domain antibodies to the same target antigen (i.e.intramolecular binding) will be highly favoured compared to binding toseparate antigens in proximity of one another (intermolecular binding,such as e.g. on a cell surface). This could, for example, reduce thechance on agonism (which might not be desired in a good therapeuticcompound). Screening and/or selection methods (as further describedherein) will allow for the isolation of avidly binding domainspositioned in relation to one another and to the antigen of interest insuch way as to have an antagonistic function only.

In another aspect of the invention, biparatopic, triparatopic ormultiparatopic polypeptides of the invention can also be selected to bepurposely agonistic. For example, a combination of two identical or twodifferent Nanobodies that bind to the Herceptin®-binding site on HER2and are genetically fused to one another can be agonistic (e.g. 2D3-2D3or 2D3 fused to other Herceptin®-competing Nanobodies). The currentinvention also provides a way to select for such agonistic biparatopic,triparatopic or multiparatopic polypeptides of the invention usingappropriate screening and/or selection procedures (as further describedherein) of members of the diversity of structural variants. Agonistscould, for example, be desired and/or interesting for triggering certainreceptors.

It is also within the scope of the invention that the linker(s) usedconfer one or more other favourable properties or functionality to thepolypeptides of the invention, and/or provide one or more sites for theformation of derivatives and/or for the attachment of functional groups(e.g. as described herein for the derivatives of the Nanobodies of theinvention). For example, linkers containing one or more charged aminoacid residues (see Table A-2 on page 48 of the International applicationWO 08/020,079) can provide improved hydrophilic properties, whereaslinkers that form or contain small epitopes or tags can be used for thepurposes of detection, identification and/or purification.

Methods for the production of a diversity of polypeptide constructsaccording to the embodiments described above, are known in the art andinclude for example the production of single domain antibodies,including dAb's, Nanobodies, V_(HH)'s and other single variable domainsand subsequently linking these single domain antibodies to each otherand optionally to other groups, moieties or binding units via suitable(peptide) linkers, such that different structural variants comprisingcombinations of the individual single domain antibodies and optionallyother groups, moieties or binding units are formed.

In this respect, naturally occurring V_(HH) domains against a particularantigen or target, can be obtained from (naïve or immune) libraries ofCamelid V_(HH) sequences. Such methods may or may not involve screeningsuch a library using said antigen or target, or at least one part,fragment, antigenic determinant or epitope thereof using one or morescreening techniques known per se. Such libraries and techniques are forexample described in WO 99/37681, WO 01/90190, WO 03/025020 and WO03/035694. Alternatively, improved synthetic or semi-synthetic librariesderived from (naïve or immune) V_(HH) libraries may be used, such asV_(HH) libraries obtained from (naïve or immune) V_(HH) libraries bytechniques such as random mutagenesis and/or CDR shuffling, as forexample described in WO 00/43507.

Yet another technique for obtaining V_(HH) sequences or Nanobodysequences directed against a particular antigen or target involvessuitably immunizing a transgenic mammal that is capable of expressingheavy chain antibodies (i.e. so as to raise an immune response and/orheavy chain antibodies directed against said antigen or target),obtaining a suitable biological sample from said transgenic mammal thatcontains (nucleic acid sequences encoding) said V_(HH) sequences orNanobody sequences (such as a blood sample, serum sample or sample ofB-cells), and then generating V_(HH) sequences directed against saidantigen or target, starting from said sample, using any suitabletechnique known per se (such as any of the methods described herein or ahybridoma technique). For example, for this purpose, the heavy chainantibody-expressing mice and the further methods and techniquesdescribed in WO 02/085945, WO 04/049794 and WO 06/008548 and Janssens etal., Proc. Natl. Acad. Sci. USA. 2006 Oct. 10; 103(41):15130-5 can beused. For example, such heavy chain antibody expressing mice can expressheavy chain antibodies with any suitable (single) variable domain, suchas (single) variable domains from natural sources (e.g. human (single)variable domains, Camelid (single) variable domains or shark (single)variable domains), as well as for example synthetic or semi-synthetic(single) variable domains.

For the generation of (single) domain antibodies from conventionalfour-chain antibodies, reference is made to EP 0 368 684, to Ward et al.(Nature 1989 Oct. 12; 341 (6242): 544-6), to Holt et al., TrendsBiotechnol., 2003, 21(11):484-490; as well as to for example WO06/030220, WO 06/003388 and other published patent applications ofDomantis Ltd.

It should also be noted that, although less preferred in the context ofthe present invention because they are not of mammalian origin, singledomain antibodies or single variable domains can be generated fromcertain species of shark (for example, the so-called “IgNAR domains”,see for example WO 05/18629).

In particular embodiments of the methods of the invention, theproduction of structural variants may typically involve generatingstructural variants with a variation with respect to the single domainantibody and/or linker (as described above), known to affect the one ormore desired characteristics. For instance, where stability is a desiredcharacteristic, the template polypeptide construct may comprise twoidentified (i.e. previously generated) single domain antibodies and thegeneration of a diversity of structural variants of the templatepolypeptide construct may involve introducing chemical modifications tothe template polypeptide construct which are envisaged to affect thehalf-life thereof (for example, by means of different forms ofpegylation); alternatively, such structural variants may comprise atleast one binding unit consisting of a single domain antibody directedagainst a serum protein (such as serum albumin), or such structuralvariants may comprise at least one additional moiety (and in particularat least one additional amino acid sequence) that increases thehalf-life of the template polypeptide construct of the invention.

Examples of structural variants which can be generated in this regardinclude but are not limited to structural variants comprising:

-   -   structural variants comprising at least two single domain        antibodies (e.g. those of the template polypeptide construct)        each suitably linked to a different poly(ethylene glycol)        polymer chain;    -   structural variants comprising at least two single domain        antibodies (e.g. those of the template polypeptide construct)        each suitably linked to one or more serum proteins or fragments        thereof (such as (human) serum albumin or suitable fragments        thereof); or    -   structural variants comprising at least one single domain        antibody linked to one or more single domain antibodies which        bind to serum proteins such as serum albumin (such as human        serum albumin), serum immunoglobulins such as IgG, or        transferrine);    -   structural variants comprising at least one single domain        antibodylinked to an Fc portion (such as a human Fe) or a        suitable part or fragment thereof, or linked to one or more        small proteins or peptides that can bind to serum proteins (such        as, without limitation, the proteins and peptides described in        WO 91/01743, WO 01/45746, WO 02/076489 and to WO 08/068,280.

In particular embodiments of methods of the present invention, thegeneration of a diversity of polypeptide constructs comprises generatingpolypeptide constructs which contain two or more binding units which aresingle domain antibodies that are directed against different antigenicdeterminants, epitopes, parts, domains, subunits or conformations (whereapplicable) of the same antigen. Generally, such polypeptide constructsbind to the antigen with increased avidity compared to a binding unitcomprising only one single domain antibody directed against the antigen.Such polypeptide constructs may for example comprise two single domainantibodies i.e. one “first” single domain antibody that is directedagainst a first antigenic determinant, epitope, part, domain, subunit orconformation (where applicable) of an antigen (which may or may not bean interaction site); and a “second” single domain antibody that isdirected against a second antigenic determinant, epitope, part, domain,subunit or conformation (where applicable) different from the firstantigenic determinant, epitope, part, domain, subunit or conformation(where applicable) of the antigen (and which again may or may not be aninteraction site). A diversity of such polypeptide constructs maycomprise combinations of different “first” and “second” single domainantibodies. Additionally or alternatively, a diversity may comprise avarying number of copies of the “first” and “second” single domainantibody. Additionally a diversity can be generated comprising more thantwo single domain antibodies directed against a different epitope (i.e.polypeptide constructs may comprise “third”, “fourth”, etc. singledomain antibodies). In particular embodiments, in such “biparatopic”and/or “multiparatopic” (as defined herein) polypeptide constructsgenerated in methods of the invention, at least one single domainantibody is directed against an interaction site (as defined herein),although the invention in its broadest sense is not limited thereto.

Accordingly, in particular embodiments, the methods of the presentinvention for obtaining a polypeptide construct directed against one ormore antigens and/or epitopes having one or more desiredcharacteristics, wherein the polypeptide construct comprises at leasttwo single domain antibodies, involve the generation of biparatopicand/or multiparatopic (such as e.g. triparatopic, tetraparatopic etc.)polypeptide constructs.

Without being limited thereto, methods for generating a diversity of bi-and mulitparatopic polypeptide constructs directed against a particularantigen and comprising at least two single domain antibodies may forexample comprise at least the step of providing a nucleic acid sequenceencoding a first amino acid sequence binding a first antigenicdeterminant, epitope, part, domain, subunit or conformation and fusingit to a set, collection or library of nucleic acid sequences encodingamino acid sequences; and ensuring adequate expression thereof.

The methods for generating a polypeptide construct of the inventiontypically further comprise screening the diversity of expressedpolypeptide constructs for constructs capable of binding to the antigenof interest with increased affinity (and/or avidity) and/or forconstructs that can bind to a second antigenic determinant, epitope,part, domain, subunit or conformation of the antigen different from theantigenic determinant, epitope, part, domain, subunit or conformationrecognized by the first amino acid sequence; optionally the methods mayinvolve screening for one or more further desired characteristics.

Further embodiments of the methods of the invention may optionallycomprise isolating the nucleic acid sequence encoding the polypeptideconstruct comprising the first amino acid sequence fused to a secondnucleic acid sequence identified in the screening step, followed byexpressing the encoded amino acid sequence.

For example, where the antigen is HER2, without being limited thereto,methods for generating a diversity of bi- and multiparatopic polypeptideconstructs directed against HER2 comprising at least two single domainantibodies may for example comprise at least the step of providing anucleic acid sequence encoding a HER2 binding amino acid sequence (suchas a single domain antibody) (binding a first antigenic determinant ofHER2) and fusing it to a set, collection or library of nucleic acidsequences encoding amino acid sequences (such as single domainantibodies); and ensuring adequate expression thereof. Accordingly, themethods of generating a polypeptide construct directed against HER2typically further comprise screening the so obtained diversity ofexpressed polypeptide constructs for constructs capable of binding toHER2 with increased affinity (and/or avidity) and/or for constructs thatcan bind to and/or have affinity for a (second) antigenic determinant ofHER2 different from the first antigenic determinant; the methods of theinvention may further optionally comprise screening for one or morefurther desired characteristics.

Further, the methods of the invention may optionally comprise isolatingthe nucleic acid sequence encoding the polypeptide construct comprisingthe HER2 amino acid sequence fused to the nucleic acid sequenceidentified in the screening step, followed by expressing the encodedamino acid sequence.

It will be understood that the nucleic acid sequences encoding thebiparatopic polypeptide constructs obtained according to the methodabove, can subsequently be fused to one or more further sets,collections or libraries of nucleic acid sequences encoding amino acidsequences and again be screened for nucleic acid sequences that encodepolypeptide constructs that can bind to and/or have affinity for anantigenic determinant on the antigen (e.g. HER2) different from thefirst and second antigenic determinants of the antigen (e.g. HER2), inorder to obtain a triparatopic or further multiparatopic polypeptideconstructs.

According to particular embodiments, methods for generating apolypeptide construct directed against HER2, which involve generatingbi- and mulitparatopic polypeptide constructs may for example compriseat least the step of:

-   -   a) providing a set, collection or library of nucleic acid        sequences, in which each nucleic acid sequence in said set,        collection or library encodes a fusion protein that comprises a        first amino acid sequence that can bind to and/or has affinity        for a first antigenic determinant, part, domain or epitope on        HER2 that is fused (optionally via a linker sequence) to a        second amino acid sequence, in which essentially each second        amino acid sequence (or most of these) is a different member of        a set, collection or library of different amino acid sequences;    -   b) screening said set, collection or library of nucleic acid        sequences for nucleic acid sequences that encode an amino acid        sequence that can bind to and/or has affinity for a second        antigenic determinant, part, domain or epitope on HER2 different        from the first antigenic determinant, part, domain or epitope on        HER-2;    -   and    -   c) isolating the nucleic acid sequences that encode an amino        acid sequence that can bind to and/or has affinity for a second        antigenic determinant, part, domain or epitope on HER2 different        from the first antigenic determinant, part, domain or epitope on        HER-2, obtained in b), optionally followed by expressing the        encoded amino acid sequence.

In these embodiments of the methods of the invention, the first aminoacid sequence in the polypeptide construct (fusion protein) encoded bysaid set collection or library of nucleic acid sequences may be the sameamino acid sequence for all members of the set, collection or library ofnucleic acid sequences encoding the polypeptide construct (fusionprotein); or the first amino acid sequence in the polypeptide construct(fusion protein) encoded by said set collection or library of nucleicacid sequences may also be a member of a set collection or library ofdifferent amino acid sequences.

In particular embodiments of methods of the invention wherein HER2 isthe antigen of interest, in step b) as described above, the set,collection or library of nucleic acid sequences may also be screened fornucleic acid sequences that encode an amino acid sequence that can bindto and/or has affinity for both a first antigenic determinant, part,domain or epitope on HER2 and a second antigenic determinant, part,domain or epitope on HER2. This may for example be performed in asubsequent steps (i.e. by in a first step screening or selecting fornucleic acid sequences that encode an amino acid sequence that can bindto and/or has affinity for the second antigenic determinant, part,domain or epitope on HER2, and subsequently in a second step selectingor screening for nucleic acid sequences that encode an amino acidsequence that can bind to and/or has affinity for the first antigenicdeterminant, part, domain or epitope on HER2; or visa versa) or in asingle step (i.e. by simultaneously screening or selecting for nucleicacid sequences that encode an amino acid sequence that can bind toand/or has affinity for both the first antigenic determinant, part,domain or epitope on HER2 and the second antigenic determinant, part,domain or epitope on HER2).

In further particular embodiments of the above-described methods, thefirst amino acid sequence used in step a) is preferably such that (i) itcan bind to and/or has affinity for the Herceptin® binding site on HER2(and may in particular be directed against domain IV of HER2, more inparticular the C-terminus of domain IV of HER2) and/or (ii) competeswith Herceptin for binding to HER-2; and in step b), the set, collectionor library of nucleic acid sequences is screened for nucleic acidsequences that encode (i) an amino acid sequence that can bind to and/orhas affinity for the Omnitarg® binding site on HER2 (and may inparticular domain II of HER2, more in particular the middle of domain IIof HER2) and/or (ii) an amino acid sequence that can compete withOmnitarg® (or the Omnitarg Fab used in Example 9) for binding to HER-2.

Alternatively, in particular embodiments, the first amino acid sequenceused in step a) is preferably such that (i) it can bind to and/or hasaffinity for the Omnitarg® binding site on HER2 (and may in particulardomain II of HER2, more in particular the middle of domain II of HER2)and/or (ii) competes with Omnitarg for binding to HER-2; and in step b),the set, collection or library of nucleic acid sequences is screened fornucleic acid sequences that encode (i) an amino acid sequence that canbind to and/or has affinity for the Herceptin® binding site on HER2 (andin particular domain IV of HER2, more in particular the C-terminus ofdomain IV of HER2) and/or (ii) an amino acid sequence that can competewith Herceptin for binding to HER-2.

In the above methods, screening or selecting for (nucleic acid sequencesthat encode) amino acid sequences that compete with Herceptin® orOmnitarg, respectively, may be performed using generally known methodsfor screening or selecting for competitors of known binding molecules,which may for example involve performing the screening or selection inthe presence of the binding molecule and/or determining the bindingaffinity of the compound(s) to be screened in the presence of thebinding molecule.

It is also possible, in step b) of the methods described above, toscreen for nucleic acid sequences that both (i) encode an amino acidsequence that can bind to and/or has affinity for the Omnitarg® bindingsite on HER2 (and in particular domain II of HER2, more in particularthe middle of domain II of HER2) and/or that can compete with Omnitarg®(or the Omnitarg Fab used in Example 9) for binding to HER-2; and thatalso (ii) encode an amino acid sequence that can bind to and/or hasaffinity for the Herceptin® binding site on HER2 (and in particulardomain IV of HER2, more in particular the C-terminus of domain IV ofHER2) and/or that can compete with Herceptin® for binding to HER-2.Again, this may be performed in separate steps or a single step, and byselecting or screening in the presence of Herceptin® and/or Omnitarg, asapplicable.

It will also be clear to the skilled person that the above methods maybe performed by screening a set, collection or library of amino acidsequences that correspond to (e.g. are encoded by) the nucleic acidsequences used in the above method; and such methods form furtheraspects of the invention.

In further particular embodiments of the methods of the invention, thestep of generating a diversity of polypeptide constructs involvesgenerating a diversity of biparatopic polypeptide constructs which arestructural variants with regard to the linker sequence, linking thesingle domain antibodies. The step of generating a diversity thus maycomprise providing a set, collection or library of nucleic acidsequences, in which each nucleic acid sequence in said set, collectionor library encodes a polypeptide construct (a fusion protein) thatcomprises a first amino acid sequence (such as a single domain antibody)that can bind to and/or has affinity for a first antigenic determinant,part, domain or epitope on an antigen of interest (such as HER2) that isfused via a linker sequence to a second amino acid sequence (such as asingle domain antibody) that can bind to and/or has affinity for asecond antigenic determinant, part, domain or epitope on the antigen ofinterest (which may be the same or different as the first antigenicdeterminant, part, domain or epitope on the antigen of interest), inwhich essentially each nucleic acid sequence (or most of these) encodesa fusion protein with a different linker sequence so as to provide aset, collection or library encoding different polypeptide constructs(fusion proteins);

The methods of the present invention for generating a polypeptideconstruct against an antigen of interest typically will further comprisethe step of screening the so obtained set, collection or library ofnucleic acid sequences for nucleic acid sequences that encode apolypeptide construct (fusion protein) that can bind to and/or hasaffinity for the first and second antigenic determinant, part, domain orepitope on the antigen of interest (e.g. HER2). Moreover, in particularembodiments, the methods of the present invention may further compriseisolating the nucleic acid sequences that encode polypeptide construct(fusion protein) that can bind to and/or has affinity for the first andsecond antigenic determinant, part, domain or epitope on the antigen ofinterest, optionally followed by expressing the encoded amino acidsequence.

As will be clear to the skilled person, these methods can be used toscreen for suitable or even optimal linker lengths for linking the firstand second amino acid sequence. For example, in this aspect, where theantigen of interest is HER2, the first amino acid sequence (such as asingle domain antibody) may be an amino acid sequence (such as a singledomain antibody and preferably a Nanobody) that can bind to and/or hasaffinity for the Omnitarg® binding site on HER2 (and may in particulardomain II of HER2, more in particular the middle of domain II of HER2)and/or that can compete with Omnitarg® (or the Omnitarg Fab used inExample 9); and the second amino acid sequence (such as a single domainantibody) may be an amino acid sequence (such as a single domainantibody and preferably a Nanobody) that can bind to and/or has affinityfor the Herceptin® binding site on HER2 (and in particular domain IV ofHER2, more in particular the C-terminus of domain IV of HER2) and/orthat can compete with Herceptin® for binding to HER-2 (or visa versa).The screening and selection may be performed as further described above.

In yet further embodiments of methods of the present invention, themethods comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding amino acid sequences (such as single domain antibodies);-   b) screening said set, collection or library of nucleic acid    sequences for a set, collection or library of nucleic acid sequences    that encode an amino acid sequence (such as a single domain    antibody) that can bind to and/or has affinity for the antigen of    interest, such as for instance HER2;-   c) ligating said set, collection or library of nucleic acid    sequences that encode an amino acid sequence (such as a single    domain antibody) that can bind to and/or has affinity for the    antigen of interest to another nucleic acid sequence that encodes an    amino acid sequence that can bind to and/or has affinity for the    antigen of interest (e.g. a nucleic acid sequence that encodes an    amino acid sequence that competes with Herceptin® for binding HER2);-   and-   d) from the set, collection or library of nucleic acid sequences    obtained in c), isolating the nucleic acid sequences encoding a    biparatopic polypeptide construct that can bind to and/or has    affinity for the antigen of interest (and e.g. further selecting for    nucleic acid sequences that encode a biparatopic amino acid sequence    that antagonizes with higher potency compared to the monovalent    amino acid sequences), followed by expressing the encoded    polypeptide construct.

The nucleic acid sequences encoding the biparatopic polypeptideconstruct obtained in the methods above, can subsequently be fused toone or more further sets, collections or libraries of nucleic acidsequences encoding amino acid sequences (such as a single domainantibodies) that can bind to and/or have affinity for the antigen ofinterest in order to obtain a triparatopic or multiparatopic amino acidsequence respectively. In addition the steps described above can also beused in the generation of polypeptide constructs directed against two(or more) different antigens (e.g. HER2 and CD3, HER2 and CD16) so as toobtain polypeptide constructs which are bispecific, trispecific ormultispecific.

Similarly, the steps described above can also be used in the generationof polypeptide constructs directed against two (or more) (different)epitopes on the same antigens as well as against two (or more) differentantigens so as to obtain polypeptide constructs which are bispecific,trispecific and/or multispecific in addition to being biparatopic,triparatopic and/or multiparatopic.

In yet further particular embodiments of the methods of the inventionfor generating a polypeptide construct directed against an antigen ofinterest (such as HER2), the methods comprise at least the steps of:

-   a) providing a first set, collection or library of nucleic acid    sequences encoding amino acid sequences (such as single domain    antibodies);-   b) screening said first set, collection or library of nucleic acid    sequences for a nucleic acid sequence that encodes an amino acid    sequence (such as a single domain antibody) that can bind to and/or    has affinity for a first antigenic determinant, part, domain or    epitope on an antigen of interest (such as HER2);-   c) ligating the nucleic acid sequence encoding said amino acid    sequence (such as a single domain antibody) that can bind to and/or    has affinity for a first antigenic determinant, part, domain or    epitope on the antigen of interest to another set, collection or    library of nucleic acid sequences encoding amino acid sequences    (such as single domain antibodies) to obtain a set, collection or    library of nucleic acid sequences that encode fusion proteins;-   d) screening said set, collection or library of nucleic acid    sequences obtained in step c) for a nucleic acid sequence that    encodes an amino acid sequence (such as a single domain antibody)    that can bind a second antigenic determinant, part, domain or    epitope on the antigen of interest different from the first    antigenic determinant, part, domain or epitope on the antigen of    interest;-   and-   e) isolating the nucleic acid sequence that encodes an amino acid    sequence (such as a single domain antibody) that can bind to and/or    has affinity for the first and second antigenic determinant, part,    domain or epitope on the antigen of interest, optionally followed by    expressing the encoded polypeptide construct.

Similar to the embodiments above, the biparatopic polypeptide constructobtained in these methods can subsequently be fused to one or morefurther sets, collections or libraries of nucleic acid sequencesencoding amino acid sequences (such as single domain antibodies) thatcan bind to and/or have affinity for the antigen of interest in order toobtain a triparatopic or multiparatopic polypeptide constructrespectively.

In addition the steps described above can also be used in the generationof polypeptide constructs directed against two (or more) differentantigens (e.g. HER2 and CD3, HER2 and CD16) so as to obtain polypeptideconstructs which are bispecific, trispecific or multispecific.

Similarly, the steps described above can also be used in the generationof polypeptide constructs directed against two (or more) (different)epitopes on the same antigens as well as against two (or more) differentantigens so as to obtain polypeptide constructs which are bispecific,trispecific and/or multispecific in addition to being biparatopic,triparatopic and/or multiparatopic.

In particular embodiments of the above method, the antigen of interestis HER2 and the first amino acid sequence (such as a single domainantibody) obtained in step b) described above is preferably such that(i) it can bind to and/or has affinity for Herceptin® binding site onHER2 (and may in particular be directed against domain IV of HER2, morein particular the C-terminus of domain IV of HER2) and/or (ii) competeswith Herceptin® for binding to HER-2; and in step d), the set,collection or library of nucleic acid sequences is screened for nucleicacid sequences that encode (i) an amino acid sequence that can bind toand/or has affinity for the Omnitarg binding site on HER2 (and may inparticular domain II of HER2, more in particular the middle of domain IIof HER2) and/or (ii) an amino acid sequence that can compete withOmnitarg (or the Omnitarg Fab used in Example 9) for binding to HER-2.

In alternative embodiments of the methods of the present invention aimedat generating polypeptide constructs against HER2, the first amino acidsequence obtained in step b) described above is preferably such that (i)it can bind to and/or has affinity for the Omnitarg binding site on HER2(and may in particular domain II of HER2, more in particular the middleof domain II of HER2) and/or (ii) competes with Omnitarg for binding toHER-2; and in step d), the set, collection or library of nucleic acidsequences is screened for nucleic acid sequences that encode (i) anamino acid sequence that can bind to and/or has affinity for theHerceptin® binding site on HER2 (and in particular domain IV of HER2,more in particular the C-terminus of domain IV of HER2) and/or (ii) anamino acid sequence that can compete with Herceptin® for binding toHER-2.

In the above methods wherein the antigen of interest is HER2 screeningor selecting for (nucleic acid sequences that encode) amino acidsequences (such as the single domain antibodies or polypeptideconstructs) that compete with Herceptin® or Omnitarg, respectively, maybe performed using generally known methods for screening or selectingfor competitors of known binding molecules, which may for exampleinvolve performing the screening or selection in the presence of thebinding molecule and/or determining the binding affinity of thecompound(s) to be screened in the presence of the binding molecule.

It is also possible, in particular embodiments of the invention aimed atgenerating suitable polypeptide constructs directed against HER2, thatstep d) as described above, encompasses screening for nucleic acidsequences that both (i) encode an amino acid sequence that can bind toand/or has affinity for the Omnitarg® binding site on HER2 (and inparticular domain II of HER2, more in particular the middle of domain IIof HER2) and/or that can compete with Omnitarg® (or the Omnitarg Fabused in Example 9) for binding to HER-2; and that also (ii) encode anamino acid sequence that can bind to and/or has affinity for theHerceptin® binding site on HER2 (and in particular domain IV of HER2,more in particular the C-terminus of domain IV of HER2) and/or that cancompete with Herceptin® for binding to HER-2. Again, this may beperformed in separate steps or a single step, and by selecting orscreening in the presence of Herceptin® and/or Omnitarg, as applicable.

In the different embodiments of the methods of the invention describedherein, the set, collection or library of nucleic acid sequencesencoding amino acid sequences may for example be a set, collection orlibrary of nucleic acid sequences encoding a naïve set, collection orlibrary of immunoglobulin sequences; a set, collection or library ofnucleic acid sequences encoding a synthetic or semi-synthetic set,collection or library of immunoglobulin sequences; and/or a set,collection or library of nucleic acid sequences encoding a set,collection or library of immunoglobulin sequences that have beensubjected to affinity maturation.

Additionally or alternatively, in the methods described herein, the set,collection or library of nucleic acid sequences may encode a set,collection or library of heavy chain variable domains (such as V_(H)domains or V_(HH) domains) or of light chain variable domains. Forexample, the set, collection or library of nucleic acid sequences mayencode a set, collection or library of domain antibodies or singledomain antibodies, or a set, collection or library of amino acidsequences that are capable of functioning as a domain antibody or singledomain antibody.

In further particular embodiments, the set, collection or library ofnucleic acid sequences may be an immune set, collection or library ofnucleic acid sequences, for example derived from a mammal that has beensuitably immunized with the antigen of interest such as HER2 or with asuitable antigenic determinant based thereon or derived therefrom, suchas an antigenic part, fragment, region, domain, loop or other epitopethereof. In one particular aspect, the antigenic determinant may be anextracellular part, region, domain, loop or other extracellularepitope(s).

The set, collection or library of nucleic acid sequences may for exampleencode an immune set, collection or library of heavy chain variabledomains or of light chain variable domains. In one specific aspect, theset, collection or library of nucleotide sequences may encode a set,collection or library of V_(HH) sequences.

In the above methods, the nucleic acid sequence encoding an amino acidsequence binding the antigen/epitope of interest fused to the set,collection or library of nucleotide sequences may be displayed on aphage, phagemid, ribosome or suitable micro-organism (such as yeast),such as to facilitate screening. Suitable methods, techniques and hostorganisms for displaying and screening (a set, collection or library of)nucleotide sequences encoding amino acid sequences (such as the singledomain antibodies or polypeptide constructs) will be clear to the personskilled in the art, for example on the basis of the further disclosureherein. Reference is also made to the review by Hoogenboom in NatureBiotechnology, 23, 9, 1105-1116 (2005).

Further reference is made to the international application of AblynxN.V. entitled “Amino acid sequences directed against HER2 andpolypeptides comprising the same for the treatment of cancers and/ortumors”, which has a filing date of Nov. 27, 2008.

Methods according to the present invention further comprise step (iii)of screening the produced diversity of polypeptide constructs of step(ii) for a polypeptide construct having one or more, more particularlytwo or more desired characteristics. As detailed above, in particularembodiments, the methods involve screening the produced diversity ofpolypeptide constructs for a polypeptide construct which, in addition toits ability to recognize the antigen/epitope of interest, has one ormore additional desired characteristics. These methods may or may notinvolve the screening for antigen-binding.

The methods of the present invention envisage obtaining a polypeptideconstruct which is a polypeptide construct (as defined herein)comprising at least two single domain antibodies (as defined herein),being directed against one or more antigens (as defined herein) and/orepitopes (as defined herein) and exhibiting one or more desiredcharacteristics. It is envisaged that the methods of the presentinvention can be used for obtaining a polypeptide construct with anydesired characteristic that can be screened for. Most typically in themethods of the invention, screening is done for one or more desiredcharacteristics selected from (but not limited to) a suitable bindingaffinity, avidity, a suitable solubility, a suitable stability, suitableefficacy, a suitable potency and/or any appropriate combinationsthereof. The suitable polypeptide construct is identified within thediversity of polypeptide constructs by screening the diversity ofpolypeptide constructs for one or more desired characteristics. Thenature of the screening step(s) in the methods described herein is thusdetermined, at least in part, by the envisaged one or more desiredcharacteristics of the polypeptide construct to be obtained.

In particular embodiments of the methods described herein, the one ormore desired characteristics include a suitable affinity for the antigenand/or epitope.

The affinity, represented by the equilibrium constant for thedissociation of an antigen with an antigen-binding protein (K_(D)), is ameasure for the binding strength between an antigenic determinant and anantigen-binding site on the antigen-binding molecule: the lesser thevalue of the K_(D), the stronger the binding strength between anantigenic determinant and the antigen-binding molecule (alternatively,the affinity can also be expressed as the affinity constant (K_(A)),which is 1/K_(D)).

The “suitable” or desired affinity of a polypeptide construct obtainedwith the methods of the invention will be determined by its intendedpurpose. In particular embodiments it is envisaged that suitableaffinity refers to the fact that the polypeptide construct binds to theone or more antigens and/or epitopes with a dissociation constant(K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to10⁻¹² moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter(i.e. with an association constant (K_(A)) of 10⁵ to 10¹² liter/moles ormore, and preferably 10⁷ to 10¹² liter/moles or more and more preferably10⁸ to 10¹² liter/moles);

and/or such that the polypeptide construct:

-   -   binds to at least one of the one or more antigens or epitopes        with a k_(on)-rate of between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹,        preferably between 10³ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, more preferably        between 10⁴ M⁻¹ s⁻¹ and 10⁷ M⁻¹ s⁻¹, such as between 10⁵ M⁻¹ s⁻¹        and 10⁷ M⁻¹ s⁻¹; and/or such that the polypeptide construct:    -   binds to at least one of the one or more antigens or epitopes        with a k_(off) rate between 1 s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹        (providing a near irreversible complex with a t_(1/2) of        multiple days), preferably between 10² s⁻¹ and 10⁻⁶ s⁻¹, more        preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴        s⁻¹ and 10⁻⁶ s⁻¹.

In particular embodiments, where the methods of the invention envisagethe generation of a polypeptide construct which is directed against twoor more antigens and/or epitopes the screening for suitable affinityinvolves the screening of the diversity of polypeptide constructs for anaffinity as described above for one, some or all of the antigens and/orepitopes against which the construct is intended to be directed.

Specific binding of an antigen-binding molecule to an antigen orantigenic determinant can be determined in any suitable manner known perse, including, for example, Scatchard analysis and/or (competitive)binding assays, such as radioimmunoassays (RIA), enzyme immunoassays(EIA) and sandwich (competition) assays, and the different variantsthereof known per se in the art; as well as the other techniquesmentioned herein.

The dissociation constant may be the actual or apparent dissociationconstant, as will be clear to the skilled person. Methods fordetermining the dissociation constant will be clear to the skilledperson, and for example include the techniques mentioned herein. In thisrespect, it will also be clear that it may not be possible to measuredissociation constants of more then 10⁻⁴ moles/liter or 10⁻³ moles/liter(e.g. of 10⁻² moles/liter). Optionally, as will also be clear to theskilled person, the (actual or apparent) dissociation constant may becalculated on the basis of the (actual or apparent) association constant(K_(A)), by means of the relationship [K_(D)=1/K_(A)].

The affinity denotes the strength or stability of a molecularinteraction. The affinity is commonly given as by the K_(D), ordissociation constant, which has units of mol/liter (or M). The affinitycan also be expressed as an association constant, K_(A), which equals1/K_(D) and has units of (mol/liter)⁻¹ (or M⁻¹). In the presentspecification, the stability of the interaction between two molecules(such as a binding unit, e.g. a single domain antibody or a polypeptideconstruct of the invention and its intended target, antigen and/orepitope) will mainly be expressed in terms of the K_(D) value of theirinteraction; it being clear to the skilled person that in view of therelation K_(A)=1/K_(D), specifying the strength of molecular interactionby its K_(D) value can also be used to calculate the corresponding K_(A)value. The K_(D)-value characterizes the strength of a molecularinteraction also in a thermodynamic sense as it is related to the freeenergy (DG) of binding by the well known relation DG=RT·ln(K_(D))(equivalently DG=−RT·ln(K_(A))), where R equals the gas constant, Tequals the absolute temperature and in denotes the natural logarithm.

The K_(D) for biological interactions which are considered meaningful(e.g. specific) are typically in the range of 10⁻¹⁰ M (0.1 nM) to 10⁻⁵ M(10000 nM). The stronger an interaction is, the lower is its K_(D).

The K_(D) can also be expressed as the ratio of the dissociation rateconstant of a complex, denoted as k_(off), to the rate of itsassociation, denoted k_(on) (so that K_(D)=k_(off)/k_(on) andK_(A)=k_(on)/k_(off)). The off-rate k_(off) has units s⁻¹ (where s isthe SI unit notation of second). The on-rate k_(on) has units M⁻¹ s⁻¹.The on-rate may vary between 10² M⁻¹ s⁻¹ to about 10⁷ M⁻¹ s⁻¹,approaching the diffusion-limited association rate constant forbimolecular interactions. The off-rate is related to the half-life of agiven molecular interaction by the relation t_(1/2)=ln(2)/k_(off). Theoff-rate may vary between 10⁻⁶ s⁻¹ (near irreversible complex with at_(1/2) of multiple days) to 1 s⁻¹ (t_(1/2)=0.69 s).

The affinity of a polypeptide construct of the invention against one ormore antigens and/or epitopes can be determined for example using thegeneral techniques for measuring K_(D). K_(A), k_(off) or k_(on). Theaffinity of a molecular interaction between two molecules can bemeasured via different techniques known per se, such as the well knownsurface plasmon resonance (SPR) biosensor technique (see for exampleOber et al., Intern. Immunology, 13, 1551-1559, 2001) where one moleculeis immobilized on the biosensor chip and the other molecule is passedover the immobilized molecule under flow conditions yielding k_(on),k_(off) measurements and hence K_(D) (or K_(A)) values. This can forexample be performed using the well-known BIACORE instruments.

It will also be clear to the skilled person that the measured K_(D) maycorrespond to the apparent K_(D) if the measuring process somehowinfluences the intrinsic binding affinity of the implied molecules forexample by artifacts related to the coating on the biosensor of onemolecule. Also, an apparent K_(D) may be measured if one moleculecontains more than one recognition sites for the other molecule. In suchsituation the measured affinity may be affected by the avidity of theinteraction by the two molecules.

Another approach that may be used to assess affinity is the 2-step ELISA(Enzyme-Linked Immunosorbent Assay) procedure of Friguet et al. (J.Immunol. Methods, 77, 305-19, 1985). This method establishes a solutionphase binding equilibrium measurement and avoids possible artefactsrelating to adsorption of one of the molecules on a support such asplastic.

However, the accurate measurement of K_(D) may be quite labour-intensiveand as consequence, often apparent K_(D) values are determined to assessthe binding strength of two molecules. It should be noted that as longas all measurements are made in a consistent way (e.g. keeping the assayconditions unchanged) apparent K_(D) measurements can be used as anapproximation of the true K_(D) and hence in the present document K_(D)and apparent K_(D) should be treated with equal importance or relevance.

Finally, it should be noted that in many situations the experiencedscientist may judge it to be convenient to determine the bindingaffinity relative to some reference molecule. For example, to assess thebinding strength between molecules A and B, one may e.g. use a referencemolecule C that is known to bind to B and that is suitably labelled witha fluorophore or chromophore group or other chemical moiety, such asbiotin for easy detection in an ELISA or FACS (Fluorescent activatedcell sorting) or other format (the fluorophore for fluorescencedetection, the chromophore for light absorption detection, the biotinfor streptavidin-mediated ELBA detection). Typically, the referencemolecule C is kept at a fixed concentration and the concentration of Ais varied for a given concentration or amount of B. As a result an IC₅₀value is obtained corresponding to the concentration of A at which thesignal measured for C in absence of A is halved. Provided K_(D ref), theK_(D) of the reference molecule, is known, as well as the totalconcentration c_(ref) of the reference molecule, the apparent K_(D) forthe interaction A-B can be obtained from following formula:K_(D)=IC₅₀/(1+c_(ref)/K_(D ref)). Note that if c_(ref)<<K_(D ref),K_(D)≈IC₅₀. Provided the measurement of the IC₅₀ is performed in aconsistent way (e.g. keeping c_(ref) fixed) for the binders that arecompared, the strength or stability of a molecular interaction can beassessed by the IC₅₀ and this measurement is judged as equivalent toK_(D) or to apparent K_(D) throughout this text.

In proteins, avidity is a term used to describe the combined strength ofmultiple bond interactions. Avidity is distinct from affinity, which isa term used to describe the strength of a single bond. As such, avidityis the combined synergistic strength of bond affinities rather than thesum of bonds. It is commonly applied to antibody interaction, wheremultiple, weak, non-covalent bonds form between antigen and antibody.Individually, each bond is quite readily broken, however when many arepresent at the same time, the overall effect results in synergistic,strong binding of antigen to antibody.

In the context of the present invention, the avidity of a polypeptideconstruct is referred to as the combined strength of bond affinities inthe complex formed between the polypeptide construct and its antigen.This combined strength of bond affinities is obtained by the binding ofeach individual single domain antibody in the polypeptide construct toits respective epitope on the antigen.

In particular embodiments, the methods of the present invention areaimed at obtaining a polypeptide construct having a suitable solubilityand the methods of the invention involve screening the diversity ofpolypeptide constructs for polypeptide constructs with a suitablesolubility. Suitable solubility values of polypeptide constructsobtainable by the methods of the present invention will be determined bytheir intended use or purpose and will be clear to the skilled personbased on the common general knowledge and the prior art as cited herein.Without being limiting, the polypeptide constructs obtained with themethod of the invention may have a solubility from 5 to 500 mg per ml,more preferably from 10 to 250 mg per ml, even more preferably from 50to 200 mg per ml, such as around 20 mg per ml, 50 mg per ml, 100 mg perml or 150 mg per ml. With regard to measuring or determining thesolubility of the polypeptide constructs obtainable according to themethods of the present invention, suitable methods are available in theart (e.g. solubility can be measured in a the dilution method, byconcentration of the polypeptide construct until precipitation of thepolypeptide construct occurs, e.g. via an ultrafiltration membrane (viacentrifugation or via crossflow filtration); or can be measuredindirectly e.g. by addition of agents such as PEG) and will be clear tothe skilled person.

In particular embodiments, the methods of the present invention areaimed at obtaining a polypeptide construct having a suitable stabilityand the methods of the invention involve screening the diversity ofpolypeptide constructs for polypeptide constructs with a suitablestability. The desired stability of a polypeptide construct will bedetermined by its intended purpose. More particularly with regard tostability, it is envisaged that a polypeptide construct may beconsidered to have a suitable stability where it has a suitablehalf-life for its intended purpose (e.g. for use as a human or animaltherapeutic).

In particular embodiments it is envisaged that suitable stability refersto the fact that the polypeptide construct has a suitable half-life. The“half-life” of a polypeptide construct of the invention can generally bedefined as the time taken for the serum concentration of the polypeptideconstruct to be reduced by 50%, in vivo, for example due to degradationof the polypeptide construct and/or clearance or sequestration of thepolypeptide construct by natural mechanisms. Suitable half-life valuesmay be a half-life that is at least 1.5 times, preferably at least 2times, such as at least 5 times, for example at least 10 times or morethan 20 times, greater than the half-life of the selected templatepolypeptide construct per se. For example, the polypeptide construct ofthe invention with increased half-life may have a half-life that isincreased with more than 1 hours, preferably more than 2 hours, morepreferably more than 6 hours, such as more than 12 hours, or even morethan 24, 48 or 72 hours, compared to the selected template polypeptideconstruct per se. For example, a polypeptide construct having a suitablestability according to the invention may have a half-life of at least 5days (such as about 5 to 10 days), preferably at least 9 days (such asabout 9 to 14 days), more preferably at least about 10 days (such asabout 10 to 15 days), or at least about 11 days (such as about 11 to 16days), more preferably at least about 12 days (such as about 12 to 18days or more), or more than 14 days (such as about 14 to 19 days).

Accordingly, in particular embodiments, the screening step of the methodaccording to the present invention encompasses determining the in vivohalf-life of the diversity of polypeptide constructs of the invention.The in vivo half-life of polypeptide construct of the invention can bedetermined in any manner known per se, such as by pharmacokineticanalysis. Suitable techniques will be clear to the person skilled in theart, and may for example generally involve the steps of suitablyadministering to a warm-blooded animal (i.e. to a human or to anothersuitable mammal, such as a mouse, rabbit, rat, pig, dog or a primate,for example monkeys from the genus Macaca (such as, and in particular,cynomologus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macacamulatta) and baboon (Papio ursinus)) a suitable dose of the polypeptideconstruct of the invention; collecting blood samples or other samplesfrom said animal; determining the level or concentration of thepolypeptide construct of the invention in said blood sample; andcalculating, from (a plot of) the data thus obtained, the time until thelevel or concentration of the amino acid sequence, compound orpolypeptide of the invention has been reduced by 50% compared to theinitial level upon dosing. Reference is for example made to theExperimental Part below, as well as to the standard handbooks, such asKenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook forPharmacists and Peters et al, Pharmacokinete analysis: A PracticalApproach (1996). Reference is also made to “Pharmacokinetics”, M Gibaldi& D Perron, published by Marcel Dekker, 2nd Rev. edition (1982). As willalso be clear to the skilled person (see for example pages 6 and 7 of WO04/003019 and in the further references cited therein), the half-lifecan be expressed using parameters such as the t1/2-alpha, t1/2-beta andthe area under the curve (AUC). In the present specification, an“increase in half-life” refers to an increase in any one of theseparameters, such as any two of these parameters, or essentially allthree these parameters. As used herein “increase in half-life” or“increased half-life” in particular refers to an increase in thet1/2-beta, either with or without an increase in the t1/2-alpha and/orthe AUC or both.

In further particular embodiments, the screening methods of the presentinvention may comprise determining certain characteristics (such as theconformation, binding, activity, molecular weight, amino acid sequence,etc.) of the polypeptide constructs after or during exposure to one ormore specific (such as e.g. denaturing condition, presence of acids,presence of basics, presence of guanidinium chloride, presence of urea,high or low temperature, high or low pressure, shear, certain time,presence of certain human tissues (such as e.g. lung tissue, livertissue, etc.) or fluids (such as e.g. saliva, mucus, BAL, blood, urine,gastric juice, etc.), etc.) conditions.

In further particular embodiments, the screening methods of the presentinvention may comprise determining the expression level of thepolypeptide constructs under specified growth and/or inducingconditions. Suitable expression levels for the polypeptide constructsare defined by their intended use or purpose and will be clear to theskilled person based on the common general knowledge and the prior artas cited herein.

In particular embodiments the methods of the present invention are aimedat generating a polypeptide construct having a suitable efficacy (in aparticular assay or model) and the methods of the invention involvescreening the diversity of polypeptide constructs to determine forpolypeptide constructs that display a suitable efficacy. A suitableefficacy value of polypeptide constructs will be determined by theintended use or purpose and will be clear to the skilled person based onthe common general knowledge and the prior art as cited herein. Withregard to measuring or determining the efficacy of the polypeptideconstructs obtainable according to the methods of the present invention,polypeptide constructs of the invention can be tested using any suitablein vitro assay, cell-based assay, in vivo assay and/or animal modelknown per se, or any combination thereof, depending on the intended useor purpose of the polypeptide construct. The selection of a suitableassay for use in the screening step will be determined by e.g. theantigen(s) and/or epitope(s) that should be bound by the polypeptideconstruct of the invention, by the specific (therapeutic) activity (suchas e.g. blocking of receptor/ligand binding; inhibition of enzymaticactivity; competing with and/or blocking a reference antibody;modulating certain signalling pathways; inducing apoptosis; etc.) thepolypeptide construct of the invention should have, etc. Based on theknowledge of the desired characteristics of the polypeptide construct ofthe invention, suitable assays and animal models will be clear to theskilled person, and for example include the assays and animal modelsused in the experimental part below and in the prior art cited herein.

In particular embodiments the methods of the present invention are aimedat generating a polypeptide construct having a suitable potency and themethods of the invention involve screening a diversity of polypeptideconstructs obtained according to methods described herein forpolypeptide constructs that display a suitable potency. Moreparticularly, suitable potency values of polypeptide constructs will bedetermined by their intended use or purpose and will be clear to theskilled person based on the common general knowledge and the prior artas cited herein. With regard to measuring or determining the potency ofthe polypeptide constructs of the present invention, these can generallybe tested using any suitable in vitro potency assay, cell-based potencyassay, in vivo potency assay and/or animal model known per se, or anycombination thereof, depending on the intended use or purpose of thepolypeptide construct. Suitable potency assays and animal models will beclear to the skilled person, and for example include the potency assaysand animal models used in the experimental part below and in the priorart cited herein.

Methods according to the present invention allow the identification,from a diversity of polypeptide constructs, of specific (candidate)polypeptide constructs having one or more desired characteristics, whichspecific (candidate) polypeptide constructs are either directed againstone antigen (whereby the polypeptide constructs may specifically bind toone or more epitopes thereof) or against different antigens (whereby thepolypeptide construct may potentially specifically hind to one or moreepitopes of each of the different antigens). Thus, the methods of thepresent invention are not limited to or defined by specific antigenicdeterminants, epitopes, parts, domains, subunits or conformations (whereapplicable) of the antigens against which the polypeptide constructs ofthe invention are directed. For example, the polypeptide constructs ofthe invention may or may not be directed against an “interaction site”(as defined herein). However, it is generally assumed and preferred thatthe polypeptide constructs of the invention are preferably directedagainst an interaction site (as defined herein).

Also, polypeptide constructs according to the invention contain one ormore binding units consisting of single domain antibodies that aredirected against one or more antigens or epitopes. Generally, suchpolypeptide constructs will bind to said one or more antigens orepitopes with increased avidity (as defined herein) compared to thebinding unit consisting of a single domain antibody. Such a polypeptideconstruct may for example comprise two single domain antibodies that aredirected against the same antigenic determinant, epitope, part, domain,subunit or conformation (where applicable) of an antigen (which may ormay not be an interaction site); or comprise at least one “first” singledomain antibody that is directed against a first antigenic determinant,epitope, part, domain, subunit or conformation (where applicable) of anantigen (which may or may not be an interaction site); and at least one“second” single domain antibody that is directed against a secondantigenic determinant, epitope, part, domain, subunit or conformation(where applicable) different from the first (and which again may or maynot be an interaction site). Preferably, in such “biparatopic”polypeptide constructs of the invention, at least one single domainantibody is directed against an interaction site (as defined herein),although the invention in its broadest sense is not limited thereto.

Also, when the antigen is part of a binding pair (for example, areceptor-ligand binding pair), the polypeptide constructs of theinvention may be such that they compete with the cognate binding partner(e.g. the ligand, receptor or other binding partner, as applicable) forbinding to the antigen, and/or such that they (fully or partially)neutralize binding of the binding partner to the antigen. Methods andassays for polypeptide constructs that compete with a cognate bindingpart and/or that neutralize binding of the binding partner will be clearto the skilled person based on the common general knowledge and theavailable prior art.

It is also within the scope of the invention that, where applicable, apolypeptide construct of the invention can bind to two or more antigenicdeterminants, epitopes, parts, domains, subunits or conformations of thesame antigen. In such a case, said antigenic determinants, epitopes,parts, domains or subunits may be essentially the same (for example, ifsaid antigen contains repeated structural motifs or occurs in amultimeric form) or may be different (and in the latter case, thepolypeptide constructs of the invention may bind to such differentantigenic determinants, epitopes, parts, domains, subunits of saidantigen with an affinity and/or specificity which may be the same ordifferent). Also, for example, when said antigen exists in an activatedconformation and in an inactive conformation, the polypeptide constructsof the invention may bind to either one of these conformations, or maybind to both these conformations (i.e. with an affinity and/orspecificity which may be the same or different). Also, for example, thepolypeptide constructs of the invention may bind to a conformation of anantigen in which it is bound to a pertinent ligand, may bind to aconformation of an antigen in which it not bound to a pertinent ligand,or may bind to both such conformations (again with an affinity and/orspecificity which may be the same or different). (all depending on thedesired characteristics of the polypeptide construct of the invention).Methods and assays for screening for polypeptide constructs that bindone or more specific conformations of an antigen will be clear to theskilled person based on the common general knowledge and the availableprior art.

It is also expected that the polypeptide constructs of the inventionwill generally bind to all naturally occurring or synthetic analogs,variants, mutants, alleles, parts and fragments of said antigen; or atleast to those analogs, variants, mutants, alleles, parts and fragmentsof said antigen that contain one or more antigenic determinants orepitopes that are essentially the same as the antigenic determinant(s)or epitope(s) to which the polypeptide constructs of the invention bindin the wild-type of said antigen. Again, in such a case, the polypeptideconstructs of the invention may bind to such analogs, variants, mutants,alleles, parts and fragments with an affinity and/or specificity thatare the same as, or that are different from (i.e. higher than or lowerthan), the affinity and specificity with which the polypeptideconstructs of the invention bind to (wild-type) antigen. It is alsoincluded within the scope of the invention that the polypeptideconstructs of the invention bind to some analogs, variants, mutants,alleles, parts and fragments of said antigen, but not to others. (alldepending on the desired characteristics of the polypeptide construct ofthe invention). Methods and assays for screening for polypeptideconstructs that bind one or more analogs, variants, mutants, alleles,parts and fragments of an antigen will be clear to the skilled personbased on the common general knowledge and the available prior art.

When said antigen exists in a monomeric form and in one or moremultimeric forms, it is within the scope of the invention that thepolypeptide constructs of the invention only bind to said antigen inmonomeric form, only bind to an antigen in multimeric form, or bind toboth the monomeric and the multimeric form. Again, in such a case, thepolypeptide constructs of the invention may bind to the monomeric formwith an affinity and/or specificity that are the same as, or that aredifferent from (i.e. higher than or lower than), the affinity andspecificity with which the polypeptide constructs of the invention bindto the multimeric form. (all depending on the desired characteristics ofthe polypeptide construct of the invention). Methods and assays forscreening for polypeptide constructs that bind one or more forms(monomeric or multimeric) of an antigen will be clear to the skilledperson based on the common general knowledge and the available priorart.

Also, when said antigen can associate with other proteins orpolypeptides to form protein complexes (e.g. with multiple subunits), itis within the scope of the invention that the polypeptide constructs ofthe invention bind to said antigen in its non-associated state, bind tosaid antigen in its associated state, or bind to both. In all thesecases, the polypeptide constructs of the invention may bind to suchmultimers or associated protein complexes with an affinity and/orspecificity that may be the same as or different from (i.e. higher thanor lower than) the affinity and/or specificity with which thepolypeptide constructs of the invention bind to said antigen in itsmonomeric and non-associated state. (all depending on the desiredcharacteristics of the polypeptide construct of the invention). Methodsand assays for screening for polypeptide constructs that bind one ormore forms (monomeric or multimeric) of an antigen will be clear to theskilled person based on the common general knowledge and the availableprior art.

In one aspect of the invention, the method described herein can be usedto screen for and so provide a polypeptide construct that is directedagainst a heterodimeric protein, polypeptide, ligand or receptor.

In this aspect of the invention, the polypeptide construct that isscreened for and so obtained will at least comprise at least a first(single) domain antibody and/or Nanobody that is directed against afirst subunit of said heterodimeric protein, polypeptide, ligand orreceptor, and at least a second (single) domain antibody and/or Nanobodythat is directed against a second subunit of said heterodimeric protein,polypeptide, ligand or receptor, different from the first subunit.

Accordingly, the diversity, collection, library or set of polypeptideconstructs that is used in the screening step (for example, thestructural variants of the selected template) will be a collection ofsuch polypeptide constructs in which each polypeptide constructcomprises a first (single) domain antibody and/or Nanobody that isdirected against a first subunit of said heterodimeric protein,polypeptide, ligand or receptor, and at least a second (single) domainantibody and/or Nanobody that is directed against a second subunit ofsaid heterodimeric protein, polypeptide, ligand or receptor.

For example, it may be that the diversity, collection, library or set ofpolypeptide constructs comprises a set of polypeptide constructs thateach share the same first (single) domain antibody and/or Nanobody (i.e.for binding to the first subunit), but in which the second (single)domain antibody (i.e. for binding to the second subunit) may differbetween the different polypeptides that form the diversity, collection,library or set (or visa versa), for example in that these second(single) domain antibodies (each) have different amino acid sequences,are humanized variants of each other, are variants of each other thathave been prepared and/or obtained through affinity maturationtechniques (and/or as part of and/or for the purposes of affinitymaturation of the original template sequence), and/or are single domainantibodies that bind to different epitopes on the second subunit; or anysuitable combination thereof.

It may also be that the diversity, collection, library or set ofpolypeptide constructs differ in both the first and/or the second(single) domain antibody, in which both the first and/or the second(single) domain antibody, respectively, that are present in a specificpolypeptide construct (i.e. that forms part of the diversity,collection, library or set that is used for screening) may again differfrom the first and/or the second (single) domain antibodies,respectively, that are present in the other polypeptides from thediversity, collection, library or set in that they have different aminoacid sequences, are humanized variants of each other, are variants ofeach other that have been prepared and/or obtained through affinitymaturation techniques (and/or as part of and/or for the purposes ofaffinity maturation of the original template sequence), and/or aresingle domain antibodies that bind to different epitopes on the firstand second subunit, respectively; or any suitable combination of theforegoing.

The heterodimeric protein, polypeptide, ligand or receptor may be anysuitable, desired and/or intended heterodimeric protein, and may forexample be a heterodimeric cytokine such as IL-12 (which consists of ap35 and a p40 subunit), IL-23 (which consists of a p19 and a p40subunit), IL-27 (which consists of an EBI-3 and p28 subunit) or IL-35(which consists of a p35 subunit and an EBI-3 subunit). Theheterodimeric protein, polypeptide, ligand or receptor may also be aheterodimeric receptor, such as a heterodimeric receptor for a(heterodimeric) cytokine, such as the cognate receptors for IL-12,IL-23, IL-27 and/or IL-35. Further reference is made to theinternational application of Ablynx N.V. entitled “Amino acid sequencesdirected against heterodimeric cytokines and/or their receptors andpolypeptides comprising the same”, which has a filing date of Nov. 27,2008. This application also contains some examples of multispecificpolypeptide constructs that comprise at least one single domain antibodyor Nanobody against a first subunit of a heterodimeric cytokine and atleast one single domain antibody or Nanobody against a second subunit ofa heterodimeric cytokine different from said first subunit (see forexample FIG. 33 for p19/p40 constructs and FIG. 36 for p35/p40constructs). It is envisaged that these and similar polypeptides may beused as templates for the methods described herein and/or that themethods described herein may be used to provide similar polypeptides(i.e. through screening of a suitable diversity, collection, library orset, as described herein).

It will also be clear that in this aspect of the invention, thediversity, collection, library or set may be screened against a group,variety, set or family of related heterodimeric proteins, polypeptides,ligands or receptors (for example, sharing a common subunit and/orbelonging to the same family, such as a family that shares similarsubunits), in order to screen for the polypeptides that have the optimalor desired specificity for one particular heterodimeric protein,polypeptide, ligand or receptor within said group, variety, set orfamily of related heterodimeric proteins, polypeptides, ligands orreceptors. As a non-limiting example, a diversity, collection, libraryor set of polypeptides of “p19/p40 constructs”, “p35/p40 constructs” orsimilar multispecific constructs that are directed against aheterodimeric cytokine or a cognate receptor for the same (as generallydescribed in the international application of Ablynx N.V. entitled“Amino acid sequences directed against heterodimeric cytokines and/ortheir receptors and polypeptides comprising the same”) may be screenedfor specificity for one of IL-12, IL-23, IL-27 and/or IL-35 compared toone or more of the other heterodimeric cytokines belonging to thisfamily (for example, p19/p40 constructs may be screened for specificityfor IL-23 compared to IL-12, IL-27 and/or IL-35. Similarly, p35/p40constructs may be screened for specificity for IL-12 compared to IL-23,IL-27 and/or IL-35). Such screening methods, and a diversity,collection, library or set that is designed for, (to be) used in and/orintended for use in such a screening method, form further aspects ofthis invention.

Also, as will be clear to the skilled person, polypeptide constructsthat contain two or more single domain antibodies directed against saidantigen may bind with higher avidity to said antigen than thecorresponding monomeric amino acid sequence(s). For example, and withoutlimitation, polypeptide constructs that contain two or more singledomain antibodies directed against different epitopes of said antigenmay (and usually will) bind with higher avidity than each of theindividual single domain antibodies, and polypeptide constructs thatcontain two or more single domain antibodies directed against saidantigen may (and usually will) bind also with higher avidity to amultimer of said antigen.

Generally, polypeptide constructs of the invention will at least bind tothose forms of said antigen (including monomeric, multimeric andassociated forms) that are the most relevant from a biological and/ortherapeutic point of view, as will be clear to the skilled person.

The present invention relates to methods for obtaining polypeptideconstructs comprising two or more single domain antibodies. Inparticular embodiments polypeptide constructs according to the inventionmay comprise at least two single domain antibodies which are selectedfrom the group of domain antibodies (or an amino acid sequence that issuitable for use as a domain antibody), “dAbs” (or an amino acidsequence that is suitable for use as a dAb), Nanobodies®, V_(HH)sequences (as defined herein, and including but not limited to a V_(HH)sequence) and/or other single variable domains or combinations thereof.For instance, a polypeptide construct according to the present inventioncomprising at least two single domain antibodies may comprise V_(H)domains and/or V_(L) domains (both derived from conventional four-chainantibodies) and/or V_(HH) domains (derived from heavy chain antibodies).It is however preferred that in a polypeptide construct of the presentinvention comprising at least two single domain antibodies, said atleast two single domain antibodies exclusively consist of only one typeof domain antibodies (i.e. corresponding to either heavy or light chaindomains). Most particularly it is envisaged that in the methods of thepresent invention polypeptide constructs are obtained wherein the atleast two single domains exclusively consist of Nanobodies® or heavychain domain antibodies (e.g. V_(H) or V_(HH)). Further particularembodiments of the invention involve methods wherein the polypeptideconstructs obtained comprise at least two single domains, whereby thesingle domains of the construct consist exclusively of (humanized)V_(HH) domains or exclusively consist of heavy chain variable domainsderived from heavy chain antibodies.

The polypeptide constructs of the invention can generally be prepared bya method which comprises at least the step of suitably linking the oneor more binding units essentially consisting of single domain antibodiesto each other and to the one or more other groups, residues, moieties orbinding units, optionally via the one or more suitable linkers, so as toprovide the polypeptide construct of the invention. Polypeptideconstructs of the invention can also be prepared by a method whichgenerally comprises at least the steps of providing a nucleic acid thatencodes a polypeptide construct of the invention, expressing saidnucleic acid in a suitable manner, and recovering the expressedpolypeptide construct of the invention. Such methods can be performed ina manner known per se, which will be clear to the skilled person, forexample on the basis of the methods and techniques further describedherein.

It will also be clear to the skilled person that the method of theinvention can equally be preformed at the nucleic acid level as well asat the amino acid level. Thus, the method of the invention can equallybe preformed by screening a diversity, set, collection or library ofnucleic acid sequences encoding a diversity, set, collection or libraryof single domain antibodies or encoding a diversity, set, collection orlibrary of polypeptide construct as well as by screening a set,collection or library of single domain antibodies or a diversity, set,collection or library of polypeptide construct.

The process of selecting and/or preparing a polypeptide construct of theinvention, starting from one or more binding units consistingessentially of single domain antibodies, is also referred to herein as“formatting” said single domain antibodies; and a single domain antibodythat is made part of a polypeptide construct of the invention is said tobe “formatted” or to be “in the format of” said polypeptide construct ofthe invention. Examples of ways in which a single domain antibody can beformatted and examples of such formats will be clear to the skilledperson based on the disclosure herein; and such formatted single domainantibodies form a further aspect of the invention.

Generally, polypeptide constructs of the invention that comprise atleast two single domain antibodies will be referred to herein as“multivalent” polypeptide constructs (as defined herein).

More specifically, a polypeptide constructs comprising at least twosingle domain antibodies that are directed against two or more differentantigens are referred to herein as “multispecific” polypeptideconstructs (as defined herein).

The methods of the present invention are directed at obtainingpolypeptide constructs comprising two or more single domain antibodies.Such a single domain antibody may be a domain antibody (or an amino acidsequence that is suitable for use as a domain antibody), a single domainantibody (or an amino acid sequence that is suitable for use as a singledomain antibody), a “dAb” (or an amino acid sequence that is suitablefor use as a dAb) or a Nanobody® (as defined herein, and including butnot limited to a V_(HH) sequence), other single variable domains, or anysuitable fragment of any one thereof.

For a general description of heavy chain antibodies and the variabledomains thereof, reference is inter alia made to the prior art citedherein, to the review article by Muyldermans in Reviews in MolecularBiotechnology 74 (2001), 277-302; as well as to the following patentapplications, which are mentioned as general background art: WO94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel;WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 ofthe Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 ofAlgonomics N.V. and Ablynx N.V.; WO 01/90190 by the National ResearchCouncil of Canada; WO 03/025020 (=EP 1 433 793) by the Institute ofAntibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the furtherpublished patent applications by Ablynx N.V. Reference is also made tothe further prior art mentioned in these applications, and in particularto the list of references mentioned on pages 41-43 of the Internationalapplication WO 06/040153, which list and references are incorporatedherein by reference.

For a general description of (single) domain antibodies, reference isfor example made to EP 0 368 684. For the term “dAb's”, reference is forexample made to Ward et al. (Nature 1989 Oct. 12; 341 (6242): 544-6), toHolt et al., Trends Biotechnol., 2003, 21(10:484-490; as well as to forexample WO 06/030220, WO 06/003388 and other published patentapplications of Domantis Ltd.

It should also be noted that, although less preferred in the context ofthe present invention because they are not of mammalian origin, (single)domain antibodies or single variable domains can be derived from certainspecies of shark (for example, the so-called “IgNAR domains”, see forexample WO 05/18629).

In particular, a (single) domain antibody may be a Nanobody® (as definedherein) or a suitable fragment thereof [Note: Nanobody®, Nanobodies® andNanoclone® are registered trademarks of Ablynx N.V.]. For a generaldescription of Nanobodies, reference is made to the further descriptionbelow, as well as to the prior art cited herein (such as WO 06/040153,WO 06/122825, WO 06/122786, WO 07/042,289, WO 07/104,529, WO 08/020,079,WO 08/074,839, WO 08/071,447, WO 08/074,840, WO 08/074,867, WO08/077,945, WO 08/101,985 by Ablynx N.V. Further reference is made tothe international application of Ablynx N.V. entitled “Amino acidsequences directed against HER2 and polypeptides comprising the same forthe treatment of cancers and/or tumors”, which has a filing date of Nov.27, 2008. In this respect, it should however be noted that thisdescription and the prior art mainly described. Nanobodies of theso-called “V_(H)3 class” (i.e. Nanobodies with a high degree of sequencehomology to human germline sequences of the V_(H)3 class such as DP-47,DP-51 or DP-29), which Nanobodies form a preferred aspect of thisinvention. It should however be noted that the invention in its broadestsense generally covers any type of Nanobody, and for example also coversthe Nanobodies belonging to the so-called “V_(H)4 class” (i.e.Nanobodies with a high degree of sequence homology to human germlinesequences of the V_(H)4 class such as DP-78), as for example describedin WO 07/118,670.

According to particular embodiment, in the methods of the inventionpolypeptide constructs are obtained comprising two or more Nanobodies orV_(HH) sequences. Generally, Nanobodies (in particular V_(HH) sequencesand partially humanized Nanobodies) can in particular be characterizedby the presence of one or more “Hallmark residues” (as described herein)in one or more of the framework sequences (again as further describedherein).

Thus, generally, a Nanobody can be defined as an amino acid sequencewith the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which one or more of the Hallmark residues are as further        defined herein.

In particular, a Nanobody can be an amino acid sequence with the(general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which the framework sequences are as further defined herein.

The amino acid residues of a Nanobody are numbered according to thegeneral numbering for V_(H) domains given by Kabat et al. (“Sequence ofproteins of immunological interest”, US Public Health Services, NIHBethesda, Md., Publication No. 91), as applied to V_(HH) domains fromCamelids in the article of Riechmann and Muyldermans, J. Immunol.Methods 2000 Jun. 23; 240 (1-2): 185-195 (see for example FIG. 2 of thispublication); or referred to herein. According to this numbering, FR1 ofa Nanobody comprises the amino acid residues at positions 1-30, CDR1 ofa Nanobody comprises the amino acid residues at positions 31-35, FR2 ofa Nanobody comprises the amino acids at positions 36-49, CDR2 of aNanobody comprises the amino acid residues at positions 50-65, FR3 of aNanobody comprises the amino acid residues at positions 66-94, CDR3 of aNanobody comprises the amino acid residues at positions 95-102, and FR4of a Nanobody comprises the amino acid residues at positions 103-113. Inthis respect, it should be noted that—as is well known in the art forV_(H) domains and for V_(HH) domains—the total number of amino acidresidues in each of the CDR's may vary and may not correspond to thetotal number of amino acid residues indicated by the Kabat numbering(that is, one or more positions according to the Kabat numbering may notbe occupied in the actual sequence, or the actual sequence may containmore amino acid residues than the number allowed for by the Kabatnumbering). This means that, generally, the numbering according to Kabatmay or may not correspond to the actual numbering of the amino acidresidues in the actual sequence. Generally, however, it can be saidthat, according to the numbering of Kabat and irrespective of the numberof amino acid residues in the CDR's, position 1 according to the Kabatnumbering corresponds to the start of FR1 and vice versa, position 36according to the Kabat numbering corresponds to the start of FR2 andvice versa, position 66 according to the Kabat numbering corresponds tothe start of FR3 and vice versa, and position 103 according to the Kabatnumbering corresponds to the start of FR4 and vice versa.

Alternative methods for numbering the amino acid residues of V_(H)domains, which methods can also be applied in an analogous manner toV_(HH) domains from Camelids and to Nanobodies, are the method describedby Chothia et al. (Nature 342, 877-883 (1989)), the so-called “AbMdefinition” and the so-called “contact definition”. However, in thepresent description, claims and figures, the numbering according toKabat as applied to V_(HH) domains by Riechmann and Muyldermans will befollowed, unless indicated otherwise.

More in particular, a Nanobody can be an amino acid sequence with the(general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4        in which FR1 to FR4 refer to framework regions 1 to 4,        respectively, and in which CDR1 to CDR3 refer to the        complementarity determining regions 1 to 3, respectively, and in        which preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-1 below;

Nanobodies may be derived in any suitable manner and from any suitablesource, and may for example be naturally occurring V_(HH) sequences(i.e. from a suitable species of Camelid) or synthetic or semi-syntheticamino acid sequences, including but not limited to “humanized” (asdefined herein) Nanobodies, “camelized” (as defined herein)immunoglobulin sequences (and in particular camelized heavy chainvariable domain sequences), as well as Nanobodies that have beenobtained by techniques such as affinity maturation (for example,starting from synthetic, random or naturally occurring immunoglobulinsequences), CDR grafting, veneering, combining fragments derived fromdifferent immunoglobulin sequences, PCR assembly using overlappingprimers, and similar techniques for engineering immunoglobulin sequenceswell known to the skilled person; or any suitable combination of any ofthe foregoing as further described herein.

Also, when a Nanobody comprises a V_(HH) sequence, said Nanobody may besuitably humanized so as to provide one or more further (partially orfully) humanized Nanobodies of the invention. Similarly, when a Nanobodycomprises a synthetic or semi-synthetic sequence (such as a partiallyhumanized sequence), said Nanobody may optionally be further suitablyhumanized, e.g. by the methods of the present invention, again so as toprovide one or more further (partially or fully) humanized Nanobodies ofthe invention.

Nanobodies of the invention can generally be obtained by any of thetechniques (1) to (8) mentioned on pages 61 and 62 of WO 08/020,079, orany other suitable technique known per se. One preferred class ofNanobodies corresponds to the V_(HH) domains of naturally occurringheavy chain antibodies. Such V_(HH) sequences can generally be generatedor obtained by suitably immunizing a species of Camelid with aparticular antigen or target (i.e. so as to raise an immune responseand/or heavy chain antibodies directed against said antigen or target),by obtaining a suitable biological sample from said Camelid (such as ablood sample, serum sample or sample of B-cells), and by generatingV_(HH) sequences directed against said antigen or target, starting fromsaid sample, using any suitable technique known per se. Such techniqueswill be clear to the skilled person and/or are described herein.

The total number of amino acid residues in a Nanobody can be in theregion of 110-120., is preferably 112-115, and is most preferably 113.It should however be noted that parts, fragments, analogs or derivatives(as further described herein) of a Nanobody are not particularly limitedas to their length and/or size, as long as such parts, fragments,analogs or derivatives meet the further requirements outlined herein andare also preferably suitable for the purposes described herein.

TABLE A-1 Hallmark Residues in Nanobodies Position Human V_(H)3 HallmarkResidues  11 L, V; L, S, V, M, W, F, T, Q, E, A, R, G, K, Y,predominantly L N, P, I; preferably L  37 V, I, F; usually V F⁽¹⁾, Y, V,L, A, H, S, I, W, C, N, G, D, T, P, preferably F⁽¹⁾ or Y  44⁽⁸⁾ G E⁽³⁾,Q⁽³⁾, G⁽²⁾, D, A, K, R, L, P, S, V, H, T, N, W, M, I; preferably G⁽²⁾,E⁽³⁾ or Q⁽³⁾; most preferably G⁽²⁾ or Q⁽³⁾.  45⁽⁸⁾ L L⁽²⁾, R⁽³⁾, P, H,F, G, Q, S, E, T, Y, C, I, D, V; preferably L⁽²⁾ or R⁽³⁾  47⁽⁸⁾ W, YF⁽¹⁾, L⁽¹⁾ or W⁽²⁾ G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D:preferably W⁽²⁾, L⁽¹⁾ or F⁽¹⁾  83 R or K; usually R R, K⁽⁵⁾, T, E⁽⁵⁾, Q,N, S, I, V, G, M, L, A, D, Y, H; preferably K or R; most preferably K 84 A, T, D; P⁽⁵⁾, S, H, L, A, V, I, T, F, D, R, Y, N, Q, predominantlyA G, E; preferably P 103 W W⁽⁴⁾, R⁽⁶⁾, G, S, K, A, M, Y, L, F, T, N, V,Q, P⁽⁶⁾, E, C; preferably W 104 G G, A, S, T, D, P, N, E, C, L;preferably G 108 L, M or T; Q, L⁽⁷⁾, R, P, E, K, S, T, M, A, H;predominantly L preferably Q or L⁽⁷⁾ Notes: ⁽¹⁾In particular, but notexclusively, in combination with KERE or KQRE at positions 43-46.⁽²⁾Usually as GLEW at positions 44-47. ⁽³⁾Usually as KERE or KQRE atpositions 43-46, e.g. as KEREL, KEREF, KQREL_(;) KQREF, KEREG, KQREW orKQREG at positions 43-47. Alternatively, also sequences such as TERE(for example TEREL), TQRE (for example TQREL), KECE (for example KECELor KECER), KQCE (for example KQCEL), RERE (for example REREG), RQRE (forexample RQREL, RQREF or RQREW), QERE (for example QEREG), QQRE, (forexample QQREW, QQREL or QQREF), KGRE (for example KGREG), KDRE (forexample KDREV) are possible. Some other possible, but less preferredsequences include for example DECKL and NVCEL. ⁽⁴⁾With both GLEW atpositions 44-47 and KERE or KQRE at positions 43-46. ⁽⁵⁾Often as KP orEP at positions 83-84 of naturally occurring V_(HH) domains. ⁽⁶⁾Inparticular, but not exclusively, in combination with GLEW at positions44-47. ⁽⁷⁾With the proviso that when positions 44-47 are GLEW, position108 is always Q in (non-humanized) V_(HH) sequences that also contain aW at 103. ⁽⁸⁾The GLEW group also contains GLEW-like sequences atpositions 44-47, such as for example GVEW, EPEW, GLER, DQEW, DLEW, GIEW,ELEW, GPEW, EWLP, GPER, GLER and ELEW.

A further detailed description of single domain antibodies, “dAbs”, andmore in particular Nanobodies®, V_(HH) sequences and/or other singlevariable domains or combinations thereof can be found on pages 15 to 36and pages 59 to 105 of WO 08/020,079, which are incorporated byreference herein.

According to a further aspect, the present invention provides for adiversity of polypeptide constructs which are structural variants of atemplate polypeptide construct.

Also, one or more or all of the polypeptide construct sequences in theabove diversity (such as set, collection or library) of structuralvariants may be obtained or defined by rational, or semi-empiricalapproaches such as computer modelling techniques or biostatics ordatamining techniques.

Furthermore, such a diversity, set, collection or library of polypeptideconstructs can comprise one, two or more sequences that are variantsfrom one another (e.g. with designed point mutations or with randomizedpositions), compromise multiple sequences derived from a diverse set ofnaturally diversified sequences (e.g. an immune library), or any othersource of diverse. Such a diversity, set, collection or library ofsequences can be displayed on the surface of a phage particle, aribosome, a bacterium, a yeast cell, a mammalian cell, and linked to thenucleotide sequence encoding the amino acid sequence within thesecarriers. This makes such a diversity amenable to selection proceduresto isolate the desired polypeptide construct in methods according to theinvention. More generally, when a sequence is displayed on a suitablehost or host cell, it is also possible (and customary) to first isolatefrom said host or host cell a nucleotide sequence that encodes thedesired sequence, and then to obtain the desired sequence by suitablyexpressing said nucleotide sequence in a suitable host organism. Again,this can be performed in any suitable manner known per se, as will beclear to the skilled person.

In particular embodiments, the diversity of polypeptide constructs is aset, collection or library of polypeptide constructs, which may be anysuitable set, collection or library of polypeptide constructs. Forexample, the set, collection or library of polypeptide constructs may bea set, collection or library of immunoglobulin sequences (as describedherein), such as a naïve or immune set, collection or library ofimmunoglobulin sequences; a synthetic or semi-synthetic set, collectionor library of immunoglobulin sequences; and/or a set, collection orlibrary of immunoglobulin sequences that have been subjected to affinitymaturation.

Also, diversity of polypeptide constructs may be a set, collection orlibrary of polypeptide constructs comprising at least two single domainantibodies that are exclusively heavy chain variable domains of heavychain antibodies (V_(HH) domains).

In a particular embodiment, the diversity, set, collection or library ofpolypeptide constructs may be an immune set, collection or library ofpolypeptide constructs, for example derived from a mammal that has beensuitably immunized with a suitable antigen or antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

In the above methods, the diversity (such as set, collection or library)of polypeptide constructs may be displayed on a phage, phagemid,ribosome or suitable micro-organism (such as yeast), such as tofacilitate screening. Suitable methods, techniques and host organismsfor displaying and screening (a set, collection or library of)polypeptide constructs will be clear to the person skilled in the art,for example on the basis of the further disclosure herein. Reference isalso made to the review by Hoogenboom in Nature Biotechnology, 23, 9,1105-1116 (2005).

According to yet a further aspect of the invention, polypeptideconstructs are provided obtainable by the methods of the presentinvention as described herein. The present invention also relates apolypeptide construct obtained by the method of the present invention.For a detailed description of such polypeptides of the invention,reference is made to the detailed description of the method of theinvention for obtaining such polypeptide constructs.

In another aspect, the invention relates to nucleic acids that encodethe polypeptide constructs obtainable by the methods of the presentinvention and to vectors comprising such nucleic acids sequences. Such anucleic acid sequence may for example be in the form of a geneticconstruct. The invention also relates to nucleic acids that encode thepolypeptide constructs obtained by the methods of the present inventionand to vectors comprising such nucleic acids sequences. Such a nucleicacid sequence may for example be in the form of a genetic construct.

In another aspect, the invention relates to a host or host cell thatexpresses (or that under suitable circumstances is capable ofexpressing) a polypeptide construct obtainable by the methods of thepresent invention; and/or that contains a nucleic acid encoding saidpolypeptide construct. The invention relates to a host or host cell thatexpresses (or that under suitable circumstances is capable ofexpressing) a polypeptide construct obtained by the methods of thepresent invention; and/or that contains a nucleic acid encoding saidpolypeptide construct.

The invention further relates to a product or composition containing orcomprising at least one polypeptide construct of the invention (or asuitable fragment thereof) and/or at least one nucleic acid encodingsaid at least one polypeptide construct of the invention, and optionallyone or more further components of such compositions known per se, i.e.depending on the intended use of the composition. Such a product orcomposition may for example be a pharmaceutical composition (asdescribed herein), a veterinary composition or a product or compositionfor diagnostic use (as also described herein).

Generally, for pharmaceutical use, the polypeptide constructs of theinvention may be formulated as a pharmaceutical preparation orcompositions comprising at least one polypeptide construct of theinvention and at least one pharmaceutically acceptable carrier, diluentor excipient and/or adjuvant, and optionally one or more furtherpharmaceutically active polypeptides and/or compounds. By means ofnon-limiting examples, such a formulation may be in a form suitable fororal administration, for parenteral administration (such as byintravenous, intramuscular or subcutaneous injection or intravenousinfusion), for topical administration, for administration by inhalation,by a skin patch, by an implant, by a suppository, etc. Such suitableadministration forms—which may be solid, semi-solid or liquid, dependingon the manner of administration—as well as methods and carriers for usein the preparation thereof, will be clear to the skilled person, and arefurther described herein.

Thus, in a further aspect, the invention relates to a pharmaceuticalcomposition that contains at least one polypeptide construct of theinvention and at least one suitable carrier, diluent or excipient (i.e.suitable for pharmaceutical use), and optionally one or more furtheractive substances.

Generally, the polypeptide constructs of the invention can be formulatedand administered in any suitable manner known per se, for whichreference is for example made to the general background art cited above(and in particular to WO 04/041862, WO 04/041863, WO 04/041865, WO04/041867 and WO 08/020,079) as well as to the standard handbooks,18^(th) Ed., Mack Publishing Company, USA (1990), Remington, the Scienceand Practice of Pharmacy, 21th Edition, Lippincott Williams and Wilkins(2005); or the Handbook of Therapeutic Antibodies (S. Dubel, Ed.),Wiley, Weinheim, 2007 (see for example pages 252-255).

For example, the polypeptide constructs of the invention may beformulated and administered in any manner known per se for conventionalantibodies and antibody fragments (including ScFv's and diabodies) andother pharmaceutically active proteins. Such formulations and methodsfor preparing the same will be clear to the skilled person, and forexample include preparations suitable for parenteral administration (forexample intravenous, intraperitoneal, subcutaneous, intramuscular,intraluminal, intra-arterial or intrathecal administration) or fortopical (i.e. transdermal or intradermal) administration.

Preparations for parenteral administration may for example be sterilesolutions, suspensions, dispersions or emulsions that are suitable forinfusion or injection. Suitable carriers or diluents for suchpreparations for example include, without limitation, those mentioned onpage 143 of WO 08/020,079. Usually, aqueous solutions or suspensionswill be preferred.

The polypeptide constructs of the invention can also be administeredusing gene therapy methods of delivery (see, e.g. U.S. Pat. No.5,399,346, which is hereby incorporated by reference in its entirety).Using a gene therapy method of delivery, primary cells transfected withthe gene encoding a polypeptide construct of the invention canadditionally be transfected with tissue specific promoters to targetspecific organs, tissue, grafts, tumors, or cells and can additionallybe transfected with signal and stabilization sequences for subcellularlylocalized expression.

Thus, the polypeptide constructs of the invention may be systemicallyadministered, e.g. orally, in combination with a pharmaceuticallyacceptable vehicle such as an inert diluent or an assimilable ediblecarrier. They may be enclosed in hard or soft shell gelatin capsules,may be compressed into tablets, or may be incorporated directly with thefood of the patient's diet. For oral therapeutic administration, thepolypeptide constructs of the invention may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 0.1% of thepolypeptide construct of the invention. Their percentage in thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of the polypeptide construct of theinvention in such therapeutically useful compositions is such that aneffective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also containbinders, excipients, disintegrating agents, lubricants and sweetening orflavouring agents, for example those mentioned on pages 143-144 of WO08/020,079. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the polypeptide constructs of the invention, sucroseor fructose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any unit dosage form should bepharmaceutically acceptable and substantially non-toxic in the amountsemployed. In addition, the polypeptide constructs of the invention maybe incorporated into sustained-release preparations and devices.

Preparations and formulations for oral administration may also beprovided with an enteric coating that will allow the constructs of theinvention to resist the gastric environment and pass into theintestines. More generally, preparations and formulations for oraladministration may be suitably formulated for delivery into any desiredpart of the gastrointestinal tract. In addition, suitable suppositoriesmay be used for delivery into the gastrointestinal tract.

The polypeptide constructs of the invention may also be administeredintravenously or intraperitoneally by infusion or injection, as furtherdescribed on pages 144 and 145 of WO 08/020,079.

For topical administration, the polypeptide constructs of the inventionmay be applied in pure form, i.e., when they are liquids. However, itwill generally be desirable to administer them to the skin ascompositions or formulations, in combination with a dermatologicallyacceptable carrier, which may be a solid or a liquid, as furtherdescribed on page 145 of WO 08/020,079.

Generally, the concentration of the polypeptide constructs of theinvention in a liquid composition, such as a lotion, will be from about0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in asemi-solid or solid composition such as a gel or a powder will be about0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

The amount of the polypeptide constructs of the invention required foruse in treatment will vary not only with the particular polypeptideconstruct selected but also with the route of administration, the natureof the condition being treated and the age and condition of the patientand will be ultimately at the discretion of the attendant physician orclinician. Also the dosage of the polypeptide constructs of theinvention varies depending on the target cell, tumor, tissue, graft, ororgan.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

An administration regimen could include long-term, daily treatment. By“long-term” is meant at least two weeks and preferably, several weeks,months, or years of duration. Necessary modifications in this dosagerange may be determined by one of ordinary skill in the art using onlyroutine experimentation given the teachings herein. See Remington'sPharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co.Easton, Pa. The dosage can also be adjusted by the individual physicianin the event of any complication.

In another aspect, the invention relates to a method for the preventionand/or treatment of at least one disease and/or disorder, said methodcomprising administering, to a subject in need thereof, apharmaceutically active amount of a polypeptide construct of theinvention and/or of a pharmaceutical composition comprising the same.

In the context of the present invention, the term “prevention and/ortreatment” not only comprises preventing and/or treating the disease,but also generally comprises preventing the onset of the disease,slowing or reversing the progress of disease, preventing or slowing theonset of one or more symptoms associated with the disease, reducingand/or alleviating one or more symptoms associated with the disease,reducing the severity and/or the duration of the disease and/or of anysymptoms associated therewith and/or preventing a further increase inthe severity of the disease and/or of any symptoms associated therewith,preventing, reducing or reversing any physiological damage caused by thedisease, and generally any pharmacological action that is beneficial tothe patient being treated.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention relates to a method for the prevention and/or treatment ofat least one disease or disorder that is associated with the one or moreantigens or epitopes against which a polypeptide construct according tothe invention is directed, with its biological or pharmacologicalactivity, and/or with the biological pathways or signalling in whichsaid one or more antigens or epitopes are involved, said methodcomprising administering, to a subject in need thereof, apharmaceutically active amount of a polypeptide construct of theinvention, of a polypeptide of the invention, and/or of a pharmaceuticalcomposition comprising the same. In particular, the invention relates toa method for the prevention and/or treatment of at least one disease ordisorder that can be treated by modulating the one or more antigens orepitopes against which a polypeptide construct according to theinvention is directed, its biological or pharmacological activity,and/or the biological pathways or signalling in which said one or moreantigens or epitopes are involved, said method comprising administering,to a subject in need thereof, a pharmaceutically active amount of apolypeptide construct of the invention, and/or of a pharmaceuticalcomposition comprising the same. In particular, said pharmaceuticallyeffective amount may be an amount that is sufficient to modulate the oneor more antigens or epitopes against which a polypeptide constructaccording to the invention is directed, its biological orpharmacological activity, and/or the biological pathways or signallingin which said one or more antigens or epitopes are involved; and/or anamount that provides a level of the polypeptide constructs of theinvention in the circulation that is sufficient to modulate the one ormore antigens or epitopes against which a polypeptide constructaccording to the invention is directed, their biological orpharmacological activity, and/or the biological pathways or signallingin which said one or more antigens are involved.

The invention furthermore relates to a method for the prevention and/ortreatment of at least one disease or disorder that can be preventedand/or treated by administering the polypeptide construct of theinvention to a patient, said method comprising administering, to asubject in need thereof, a pharmaceutically active amount of apolypeptide construct of the invention, and/or of a pharmaceuticalcomposition comprising the same.

More in particular, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder chosen from thegroup consisting of the diseases and disorders listed herein, saidmethod comprising administering, to a subject in need thereof, apharmaceutically active amount of a polypeptide construct of theinvention, and/or of a pharmaceutical composition comprising the same.

In another aspect, the invention relates to a method for immunotherapy,and in particular for passive immunotherapy, which method comprisesadministering, to a subject suffering from or at risk of the diseasesand disorders mentioned herein, a pharmaceutically active amount of apolypeptide construct of the invention and/or of a pharmaceuticalcomposition comprising the same.

In the above methods, the polypeptide constructs of the invention and/orthe compositions comprising the same can be administered in any suitablemanner, depending on the specific pharmaceutical formulation orcomposition to be used. Thus, the polypeptide constructs of theinvention and/or the compositions comprising the same can for example beadministered orally, intraperitoneally (e.g. intravenously,subcutaneously, intramuscularly, or via any other route ofadministration that circumvents the gastrointestinal tract),intranasally, transdermally, topically, by means of a suppository, byinhalation, again depending on the specific pharmaceutical formulationor composition to be used. The clinician will be able to select asuitable route of administration and a suitable pharmaceuticalformulation or composition to be used in such administration, dependingon the disease or disorder to be prevented or treated and other factorswell known to the clinician.

The polypeptide constructs of the invention and/or the compositionscomprising the same are administered according to a regime of treatmentthat is suitable for preventing and/or treating the disease or disorderto be prevented or treated. The clinician will generally be able todetermine a suitable treatment regimen, depending on factors such as thedisease or disorder to be prevented or treated, the severity of thedisease to be treated and/or the severity of the symptoms thereof, thespecific polypeptide construct of the invention to be used, the specificroute of administration and pharmaceutical formulation or composition tobe used, the age, gender, weight, diet, general condition of thepatient, and similar factors well known to the clinician.

Generally, the treatment regimen will comprise the administration of oneor more polypeptide constructs of the invention, or of one or morecompositions comprising the same, in one or more pharmaceuticallyeffective amounts or doses. The specific amounts) or doses toadministered can be determined by the clinician, again based on thefactors cited above.

Generally, for the prevention and/or treatment of the diseases anddisorders mentioned herein and depending on the specific disease ordisorder to be treated, the potency of the specific polypeptideconstruct of the invention to be used, the specific route ofadministration and the specific pharmaceutical formulation orcomposition used, the polypeptide constructs of the invention willgenerally be administered in an amount between 1 gram and 0.01 microgramper kg body weight per day, preferably between 0.1 gram and 0.1microgram per kg body weight per day, such as about 1, 10, 100 or 1000microgram per kg body weight per day, either continuously (e.g. byinfusion), as a single daily dose or as multiple divided doses duringthe day. The clinician will generally be able to determine a suitabledaily dose, depending on the factors mentioned herein. It will also beclear that in specific cases, the clinician may choose to deviate fromthese amounts, for example on the basis of the factors cited above andhis expert judgment. Generally, some guidance on the amounts to beadministered can be obtained from the amounts usually administered forcomparable conventional antibodies or antibody fragments against thesame target administered via essentially the same route, taking intoaccount however differences in affinity/avidity, efficacy,biodistribution, half-life and similar factors well known to the skilledperson.

Usually, in the above method, a single polypeptide construct of theinvention will be used. It is however within the scope of the inventionto use two or more polypeptide constructs of the invention incombination.

The polypeptide constructs of the invention may also be used incombination with one or more further pharmaceutically active compoundsor principles, i.e. as a combined treatment regimen, which may or maynot lead to a synergistic effect. Again, the clinician will be able toselect such further compounds or principles, as well as a suitablecombined treatment regimen, based on the factors cited above and hisexpert judgement.

In particular, the polypeptide constructs of the invention may be usedin combination with other pharmaceutically active compounds orprinciples that are or can be used for the prevention and/or treatmentof the diseases and disorders cited herein, as a result of which asynergistic effect may or may not be obtained. Examples of suchcompounds and principles, as well as routes, methods and pharmaceuticalformulations or compositions for administering them will be clear to theclinician.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are to be administered simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition, as will be clear tothe skilled person.

Also, when two or more active substances or principles are to be used aspart of a combined treatment regimen, each of the substances orprinciples may be administered in the same amount and according to thesame regimen as used when the compound or principle is used on its own,and such combined use may or may not lead to a synergistic effect.However, when the combined use of the two or more active substances orprinciples leads to a synergistic effect, it may also be possible toreduce the amount of one, more or all of the substances or principles tobe administered, while still achieving the desired therapeutic action.This may for example be useful for avoiding, limiting or reducing anyunwanted side-effects that are associated with the use of one or more ofthe substances or principles when they are used in their usual amounts,while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to theinvention may be determined and/or followed in any manner known per sefor the disease or disorder involved, as will be clear to the clinician.The clinician will also be able, where appropriate and on a case-by-casebasis, to change or modify a particular treatment regimen, so as toachieve the desired therapeutic effect, to avoid, limit or reduceunwanted side-effects, and/or to achieve an appropriate balance betweenachieving the desired therapeutic effect on the one hand and avoiding,limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desiredtherapeutic effect is achieved and/or for as long as the desiredtherapeutic effect is to be maintained. Again, this can be determined bythe clinician.

In another aspect, the invention relates to the use of a polypeptideconstruct of the invention in the preparation of a pharmaceuticalcomposition for prevention and/or treatment of at least one diseaseand/or disorder; and/or for use in one or more of the methods oftreatment mentioned herein.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention also relates to the use of a polypeptide construct of theinvention in the preparation of a pharmaceutical composition for theprevention and/or treatment of at least one disease or disorder that canbe prevented and/or treated by administering a polypeptide construct ofthe invention to a patient.

More in particular, the invention relates to the use of a polypeptideconstruct of the invention in the preparation of a pharmaceuticalcomposition for the prevention and/or treatment of diseases and/ordisorders, and in particular for the prevention and treatment of one ormore of the diseases and disorders listed herein.

Again, in such a pharmaceutical composition, the one or more polypeptideconstructs of the invention may also be suitably combined with one ormore other active principles, such as those mentioned herein.

The invention will now be further described by means of the followingnon-limiting preferred aspects, examples and figures:

FIGURE LEGENDS

FIG. 1: Anti-HER2 humoral immune response induced after immunisation ofLlama glama with HER2-overexpressing SKBR3 cells. The reactivity ofpre-immune (day 0) and immune sera (day 42 and day of PBL1 take) ofanimals 121 and 122 immunized with whole cells was determined by ELISAusing rhErbB2-Fc as antigen according to particular embodiments of theinvention (e.g. see Example 3.2). A: total IgG response; B: IgG1 isotyperesponse; C: IgG2 isotype response; D: IgG3 isotype response.

FIG. 2: HER2-specific ELISA analysis of periplasmic preparationscontaining myc-tagged Nanobody protein fragments from selected clones,according to particular embodiments of the invention. Periplasmicpreparations of soluble Nanobody protein fragments were added to wellsof an ELISA plate, which had been coated with rhErbB2/Fc antigen and hadbeen additionally blocked with PBS+1% casein. Detection was performed bya monoclonal anti-myc antibody followed by an alkalinephosphatase-conjugated polyclonal goat anti-mouse antibody. The ELISAwas developed by a PNPP-substrate as described in Example 6. TheOD-values (Y-axis) were measured at 405 nm by an ELISA-reader. Each barrepresents an individual periplasmic extract.

FIG. 3: Flow cytometric analysis of selected clones (2A1, 2A4, 2C3, 2C5,2D3 and 2G4) according to particular embodiments of the invention.Nanobody-containing periplasmic extracts were added to ErbB2overexpressing SKBR3 cells. Detection was performed by a monoclonalanti-myc antibody followed by a PE-labeled polyclonal anti-mouseantibody. Nanobodies binding to cells was measured by an increase influorescence intensity as compared to cells that were incubated withFACS buffer (PBS+10% FBS) followed by monoclonal anti-myc antibody andPE-labeled polyclonal anti-mouse antibody. Fluorescence intensity isblotted on the X-axis, the number of events on the Y-axis.

FIG. 4: Herceptin® competitive ELISA according to particular embodimentsof the invention. An ELISA plate was coated with SKBR3 vesicles (5μg/ml) and additionally blocked with PBS+1% casein. 2 nM Herceptin® wasadded to the wells, after which periplasmic preparations of solubleNanobody protein fragments were added. Detection of Herceptin® bindingto SKBR3 vesicles was performed by an alkaline phosphatase-conjugatedAffiniPure Goat Anti-Human IgG, Fc Fragment Specific (JacksonImmunoResearch Labs, Suffolk, UK). The ELISA was developed by aPNPP-substrate as described in Example 6. The OD-values (Y-axis) weremeasured at 405 nm by an ELISA-reader. Each bar represents an individualperiplasmic extract. The OD value corresponding to the maximal signalrepresents the OD value measured for binding of Herceptin® withoutaddition of periplasmic extract containing HER-binding Nanobody. Theminimal signal represents the background staining of non-coated wellsincubated only with alkaline phosphatase-conjugated AffiniPure GoatAnti-Human IgG, followed by detection using a PNPP-substrate. Controls1-5 represent individual periplasmic extracts containing non-HER2binding Nanobodies.

FIG. 5: Herceptin®-competitive FMAT according to particular embodimentsof the invention. Dilutions of periplasmic extracts containing HER2binding Nanobodies were tested for their ability to block the binding ofHerceptin® to HER2-overexpressing SKBR3 cells as described in Example 8.(−) represents the signal obtained for binding of Alexa647-labeledHerceptin® without addition of periplasmic extract. Addition ofperiplasmic extract containing a non-HER2 binding Nanobody (irr) had noinfluence on the binding of Herceptin® to SKBR3 cells. Periplasmicextracts 2A4, 2A5, 2A6, 2B1, 2B2, 2B4, 2B5, 2C1, 2C3, 2D2 and 2D3blocked binding of Herceptin® to HER2 with more than 80%.

FIG. 6: Herceptin®-competitive FMAT analysis according to particularembodiments of the invention. Nanobodies compete with binding ofHerceptin® to SKBR3 cells in a dose-dependent manner as described inExample 8.

FIG. 7: Omnitarg-Fab competitive FMAT according to particularembodiments of the invention. Dilutions of periplasmic extractscontaining HER2 binding Nanobodies were tested for their ability toblock the binding of Omnitarg-Fab (OT-Fab) to HER2-overexpressing SKBR3cells as described in Example 9. (cells) represents the signal obtainedfor binding of biotinylated OT-Fab without addition of periplasmicextract. Addition of periplasmic extract containing a non-HER2 bindingNanobody (irr) had no influence on the binding of OT-Fab to SKBR3 cells.Periplasmic extracts 47A8, 47A11, 47B1, 47B12, 47D1, 47D4, 47D5, 47E7,47F5 and 47G7 blocked binding of OT-Fab to HER2 with more than 85%.

FIG. 8: Growth inhibitory effect of monovalent HER2 binding Nanobodieson ErbB2-overexpressing SKBR3 cells according to particular embodimentsof the invention. SKBR3 cells were seeded in 96 well plates and allowedto adhere as explained in Example 11. HER2-binding Nanobodies 5F7, 2A5,2A4, 2D3 and 2C3, non-HER2 binding irrelevant Nanobody 12B2 or mediumalone were added and the cells were incubated for 3 days. During thelast 24 h, cells were pulsed with 1 μCi [³H]-thymidine. Incorporation of[³H]-thymidine was measured as described in Example 11.

FIG. 9: Herceptin®-competitive FMAT. Dilutions of monovalent, bivalentand bispecific Nanobodies were tested for their ability to block thebinding of Herceptin® to HER2-overexpressing SKBR3 cells according toparticular embodiments of the invention (e.g. as described in Example12). Bispecific Nanobodies 2A4-9GS-ALB1 and 2A5-9GS-ALB1 blocked thebinding of Herceptin® to HER2-expressing SKBR3 cells to the same extentas the monovalent 2A4 and 2A5 Nanobodies respectively. Bivalent2A4-9GS-2A4 and 2A5-9GS-2A5 Nanobodies blocked the binding of Herceptin®to HER2-expressing SKBR3 cells to a greater extent than their monovalentformat. A: 2A4 derivatives; B: 2A5 derivatives.

FIG. 10: Design of biparatopic Nanobody expression vector according toparticular embodiments of the invention (e.g. as described in Example13.1).

FIG. 11: SKBR3 cell proliferation assay with biparatopic Nanobodiespurified from periplasmic extracts derived from plate 27 by PhyTip200⁺according to particular embodiments of the invention. BiparatopicNanobodies 27A2-35GS-2D3, 27A5-35GS-2D3, 27B3-35GS-2D3, 27B5-35GS-2D3,27C4-35GS-2D3, 27D3-35GS-2D3 and 27D6-35GS-2D3 block SKBR3 cellproliferation to a greater extent than 50 nM Herceptin®. BiparatopieNanobodies 27A7-35GS-2D3, 27A9-35GS-2D3, 27A1′-35GS-2D3, 27A12-350S-2D3,27B11-35GS-2D3, 27C11-350S-2D3 and 27D7-35GS-2D3 display an agonisticeffect.

FIG. 12: Sensorgram of monovalent 2D3, bivalent 2D3-35GS-2D3 anddummy-2D3 biparatopic Nanobodies according to particular embodiments ofthe invention.

FIG. 13: Sensorgram of monovalent 2D3 and biparatopic Nanobodies27B7-35GS-2D3, 27C3-35GS-2D3 and 27H5-35GS-2D3 according to particularembodiments of the invention.

FIG. 14: Sensorgram of monovalent 2D3, bivalent 2D3-35GS-2D3 andbiparatopic Nanobodies 27A3-35GS-2D3, 27E7-35GS-2D3 and 27D1-35GS-2D3according to particular embodiments of the invention.

FIG. 15: Herceptin®-competitive FMAT. Dilutions of monovalent 2D3,bivalent 2D3-35GS-2D3 and biparatopic Nanobodies combining theHerceptin®-competitive 2D3 and a HER2-binding or dummy Nanobody weretested for their ability to block the binding of Herceptin® toHER2-overexpressing SKBR3 cells according to particular embodiments ofthe invention (e.g. as described in Example 14.2). A: Bivalent2D3-35GS-2D3 and biparatopic Nanobodies 27H3-35GS-2D3 and 27D1-35GS-2D3block binding of Herceptin® to HER2 expressed on SKBR3 cells moreefficiently than monovalent 2D3 Nanobody. B: Nanobodies 27A3, 27A5 and30D10 have no influence on the Herceptin®-competitive behavior ofNanobody 2D3 when fused to its N-terminal end, spaced by a 35GS linker.C: Nanobodies 27B7, 27C3, 27H5 and the dummy Nanobody have an inhibitoryeffect on the Herceptin®-competitive potential of 2D3 when fused to itsN-terminal end, spaced by a 35GS linker.

FIG. 16: Omnitarg-Fab competitive FMAT according to particularembodiments of the invention. Dilutions of OT-Fab, monovalent 2D3 andbiparatopic Nanobodies 27C3-35GS-2D3, 27A5-35GS-2D3, 27H3-35GS-2D3 anddummy-35GS-2D3 were tested for their ability to block the binding ofOT-Fab to HER2-overexpressing SKBR3 cells as described in Example 14.3.None of the bipartope Nanobodies, nor monovalent 2D3 blocked the bindingof OT-Fab to HER2 expressed on SKBR3 cells. OT-Fab blocked binding ofbiotinylated OT-Fab in a dose-dependent manner.

FIG. 17: Effect of biparatopic Nanobodies on SKBR3 tumor cellproliferation according to particular embodiments of the invention.Biparatopic Nanobodies 27A5-35GS-2D3, 27A3-35GS-2D3 and 30D10-35GS-2D3significantly block proliferation of SKBR3 tumor cells and to a greaterextent than the monovalent 2D3 and dummy-2D3 biparatopic Nanobody.

FIG. 18: Effect of biparatopic Nanobodies on AKT signaling in SKBR3cells according to particular embodiments of the invention (e.g. seeExample 16). Biparatopic 27A3-35GS-2D3 and 27A5-35GS-2D3, Herceptin®,but not dummy-2D3 biparatopic or monovalent 2D3 Nanobody inhibits AKTphosphorylation in whole SKBR3 cell lysates.

FIG. 19: Sensorgram of HER2-ECD binding to 2D3, 47D5 or the biparatopicNanobody 2D3-35GS-47D5 according to particular embodiments of theinvention.

FIG. 20: Effect of biparatopic Nanobodies combiningHerceptin®-competitive and Omnitarg competitive Nanobodies, monovalentNanobodies 2D3, 5F7 and 47D5, Omnitarg-Fab and Herceptin® onHRG-mediated activation of mitogen-activated protein kinase (MAPK)according to particular embodiments of the invention.

FIG. 21: Effect of biparatopic Nanobodies combiningHerceptin®-competitive and Omnitarg competitive Nanobodies, monovalentNanobodies 2D3, 5F7 and 47D5, Omnitarg-Fab and Herceptin® onHRG-mediated activation of Akt signaling according to particularembodiments of the invention.

FIGS. 22A and 22B: Model of NB-2D3 (blue) linked to another Nanobody(cyan) docked on HER-2 (red) according to particular embodiments of theinvention. The linker is shown in black. N denotes the N-terminus ofNB-2D3; C is the C-terminus of Nb-2D3.

FIG. 23: Energy penalty values for each residue in the linker +/−10residues of each Nanobody connected to the linker in the biparatopicconstruct 5F7-35GS-47D5 with appropriate linker length according toparticular embodiments of the invention. None of the residues of thelinker or at the connection points of the linker with the Nanobodies(NB-1 and NB-2) have a high energy penalty value.

FIG. 24: Energy penalty values for each residue in the linker +/−10residues of each Nanobody connected to the linker in the biparatopicconstruct 47D5-35GS-5F7 with unappropriate linker length according toparticular embodiments of the invention. High energy penalty values areobserved at the C-terminal connection of the linker with the N-terminalend of the second Nanobody (NB-2).

FIG. 25: Energy penalty values for each residue in the linker +/−10residues of each Nanobody connected to the linker in a biparatopicconstruct with the same Nanobodies as in FIG. 23 but with a longerlinker length (47D5-40GS-5F7) according to particular embodiments of theinvention. We see that the high energy penalty values at the connectionof the C-terminal end of the linker with the N-terminal end of NB-2 arereduced suggesting a more appropriate linker length. The energy penaltyvalues at both ends of the linker are still higher than those observedin FIG. 22, indicating a still not optimal linker.

FIG. 26A: Backbone RMSD (Å²) between the 5F7-linker-47D5 constructs(built by homology modelling) with the individual Nanobodies 5F7 and47D5 in their unlinked binding mode according to particular embodimentsof the invention. The linker length varies from 5 to 35.

FIG. 26B: Ribbon view of the 5F7-linker-47D5 biparatopic construct for 2linker lengths according to particular embodiments of the invention. Thebinding mode of the individual Nanobodies is shown in blue; thebiparatopic constructs are in red. The HER-2 target is omitted forclarity. On the left side: a 35GS linker is used between the 2Nanobodies and a very limited deviation from the individual bindingmodes is observed. On the right side: a 5GS linker is used and it canclearly been observed that both Nanobodies in the biparatopic constructsignificantly deviate from their optimal binding mode.

FIGS. 27A-K: Figures illustrating some of the preferred aspects and someof the advantages of the present invention, including the multiparatopicpolypeptides of the invention.

EXAMPLES Example 1 Procurement of the Extracellular Domain of HER2 foruse as Selection Antigen in Phage Display 1.1 Cloning of ExtracellularHER2 Domain

cDNA was isolated from SKBR3 breast cancer cells. The isolation of totalRNA and cDNA synthesis was done according to standard protocols(Sambrook, Molecular cloning: Laboratory manual, 2^(nd) edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Thecoding sequence of the extracellular domain of the HER2 antigen wasamplified by PCR using primer For-ErbB2 ECD; GCGAGCACCCAAGTGTGCACC (SEQID NO: 2267) and primer Rev-ErbB2 ECD: CTGCTCGGCGGGGCAGCCCTT (SEQ ID NO:2268). The PCR construct was then cloned into the pCR4-TOPO cloningvector (Invitrogen, Paisley, UK). Clone 4 having the correct sequencewas then amplified by PCR using primers (For-pST ErbB2 ECD:GGCGCGCCGACTACAAAGACGATGACGACAAGAGCACCCAAGTGTGCACC (SEQ ID NO: 2269) andRev-pST ErbB2 ECD: CGGCTCGAGCTATTAATGAGAATGGTGATGGTGCTCGGCGGGGCAGCCCTT(SEQ ID NO: 2270)) that were designed to introduce restriction sites atthe beginning and the end of the fragment encoding the HER2-ECD. The PCRproduct was then cloned via AscI and XhoI into the plasmidpSecTag-HygroA (Invitrogen, Paisley, UK). As such, the coding sequenceof the HER2-ECD was fused in frame with the Ig-κ chain leader sequenceat its N-terminal end followed by a Flag tag and a polyhistidine tag atthe C-terminus. The sequence of different clones was determined bysequencing according to standard protocols.

1.2 Expression of the Extracellular Domain of HER2 in HEK 293 Cells,Purification of the Recombinant Protein

Expression of the extracellular domain of HER2 was performed in HEK293Tcells. HEK293T cells were seeded at 2×10⁶ cells in 20 ml Dulbecco'sModified Eagle's Medium (DMEM) containing 10% FBS in T75 tissue cultureflasks and allowed to adhere overnight. The next day, culturesupernatant was removed and the cells were transiently transfected withpurified pSecTag-HygroA plasmid DNA using Fugene-HD (Roche, Basel,Switzerland) as transfection agent. Cells were grown for an additional72 h in DMEM containing 0.1% FBS, after which the culture supernatantwas collected and filter-sterilized on a 0.22 μm filter (Millipore). Theconstruct was then further purified out of the culture supernatant byimmobilized metal affinity chromatography (IMAC) and size exclusionchromotagraphy (SEC).

Detection of the recombinant protein was performed by ELISA. Maxisorp96-well plate (Num, Wiesbaden, Germany) was coated with an anti-flagmonoclonal antibody (Sigma Aldrich, Bornem, Belgium). Unspecific bindingwas blocked with 2% milk powder in PBS for 2 hours. All prior andsubsequent washes were performed with PBS. Afterward, eluate fractionswere incubated for 2 hours at room temperature, followed by incubationwith Herceptin®. Detection of the recombinant HER2-ECD was performedwith a horseradish peroxidase conjugated anti-IgG antibody (JacksonImmunoresearch Laboratories, Suffolk, UK). Development of the ELISA wasperformed with TMB substrate (Pierce, Rockford, Ill.) according to thespecifications of the manufacturer

Example 2 Procurement of Omnitarg-Fab for use as Competitive Agent inPhage Display and Screening Assays 2.1 Cloning of Omnitarg-Fab

Omnitarg-Fab was constructed by gene assembly. The amino acid sequenceof variable light and variable heavy chain of Omnitarg was derived frompatents WO 2006/044908 and WO 2004/048525. The sequence wasbacktranslated and codon optimized using Leto 1.0 Gene optimizationsoftware (www.entechelon.com). Oligonucleotide primers for assembly ofthe variable light chain (V_(L)), variable heavy chain (V_(H)), constantlight chain (C_(L)) and constant domain 1 of the heavy chain (CH₁) ofthe Omnitarg-Fab were designed (Tables C-5 and C-6) and assembly PCRperformed. The introduced restriction sites SfiI and BsiWI for theV_(L). KpnI and BstEII for the V_(H), BsiWI and AscI for the C_(L), andBstEII and NotI for the CH₁ were utilized for sequential cloning into anin-house expression vector derived from pUC119 which contained the LacZpromoter, a resistance gene for ampicillin or carbenicillin, amulticloning site and the gen3 leader sequence. In frame with theOmnitarg-Fab coding sequence, the vector coded for a C-terminal c-myctag and a (His)6 tag. Oligonucleotide sequences were designed to have a15 nucleotide overlap with 5′ and 3′ overlapping oligonucleotides. Threeconsecutive PCR overlap extension rounds were performed using ExpandHigh fidelity PCR system (Roche, Basel, Switzerland) to obtain V_(L),V_(H), C_(L) and CH₁ respectively. The obtained PCR fragments werecloned into the pCR4-TOPO cloning vector (Invitrogen, Paisley, UK).Plasmid DNA was prepared from clones having the correct sequence. Thefragments were isolated from the pCR4-TOPO cloning vector viarestriction with the appropriate enzymes and extraction of the fragmentsfrom agarose gel. The fragments were then consecutively cloned into thein-house expression vector.

2.2 Expression of the Omnitarg-Fab in E. coli Cells, Purification of theRecombinant Protein

The Omnitarg-Fab fragment was expressed in E. coli as His6-taggedprotein and subsequently purified from the culture medium by immobilizedmetal affinity chromatography (IMAC) and size exclusion chromatography(SEC).

Omnitarg-Fab was biotinylated using EZ-Link Sulpha-NHS-LC-Biotinlabeling kit according to the manufacturer's instructions (Pierce,Rockford, Ill.). Removal of free biotin was performed on Zeba DesaltSpin columns according to the manufacturer's instructions (Pierce,Rockford, Ill.).

Example 3 Identification of HER2 Binding Nanobodies 3.1 Immunizations

After approval of the Ethical Committee of the Faculty of VeterinaryMedicine (University Ghent, Belgium), 2 llamas (121, 122) wereimmunized, according to standard protocols, with 6 intramuscularinjections at biweekly intervals of SKBR3 human tumor cells which arederived from a breast tumor and contain an amplified HER2 gene andoverexpress HER2 p185 tyrosine kinase (SKBR3; ATCC HTB-30; LGCPromochem, Middlesex, UK). Each dose consisted of approximately 5×10⁷freshly harvested SKBR3 cells.

3.2 Evaluation of Induced Responses in Llama

At day 0, 42 and 81 (time of PBL collection), sera were collected toevaluate the induction of immune responses in the animals against HER2by ELISA. In short, 2 μg/ml recombinant human ErbB2/Fc chimera(rhErb2-Fc; R&D Systems, Minneapolis, Minn.) were immobilized overnightat 4° C. in a 96 well Maxisorp plate (Num, Wiesbaden, Germany). Wellswere blocked with a casein solution (1% in PBS). After addition of serumdilutions, specifically bound immunoglobulins were detected using a goatanti-llama horseradish peroxidase conjugate (Bethyl Lab. Inc.,Montgomery, Tex.), showing that for all animals a significant antibodydependent immune response against HER2 was induced (FIG. 1A). Theantibody response was mounted both by the conventional and the heavychain only antibody expressing B-cell repertoires since specificallybound immunoglobulins could be detected with antibodies specificallyrecognizing the conventional llama IgG1 antibodies (FIG. 1B) or theheavy-chain only llama IgG2 (FIG. 1C) and IgG3 (FIG. 1D) antibodies.

3.3 Library Construction

When an appropriate immune response was induced in llama, four daysafter the last antigen injection, a 150 ml blood sample was collectedand peripheral blood lymphocytes (PBLs) were purified by a densitygradient centrifugation on Ficoll-Paque™ (Amersham Biosciences, Uppsala,Sweden) according to the manufacturer's instructions. Next, total RNAwas extracted from these cells and used as starting material for RT-PCRto amplify Nanobody encoding gene fragments. These fragments were clonedinto a phagemid vector derived from pUC119 which contained the LacZpromoter, a coliphage pIII protein coding sequence, a resistance genefor ampicillin or carbenicillin, a multicloning site and the gen3 leadersequence. In frame with the Nanobody® coding sequence, the vector codedfor a C-terminal c-myc tag and a (His)6 tag. Phage was preparedaccording to standard methods (see for example the prior art andapplications filed by applicant cited herein) and stored after filtersterilization at 4° C. for further use.

3.4 Selections

Phage libraries obtained from llamas 121 and 122 were used for differentselections.

In a first selection, ErbB2/Fc chimera (R&D Systems, Minneapolis, Minn.,US) was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany)at 20, 5 and 1 nM. Following incubation with the phage libraries andextensive washing, bound phage was specifically eluted with trypsin (1mg/ml).

In a second selection. ErbB2/Fc chimera (R&D Systems, Minneapolis, US)was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany) at 20nM. Following incubation with the phage libraries and extensive washing,bound phage was specifically eluted with Herceptin® (Genentech, Roche).

In a third selection, soluble biotinylated ErbB2/Fc chimera wasincubated with the phage libraries. After extensive washing, thebiotinylated ErbB2/Fc was captured on a neutravidin coated solid phase.Bound phage was specifically eluted with trypsin (1 mg/ml).

In a fourth selection, soluble biotinylated ErbB2/Fc chimera wasincubated with the phage libraries. After adding a 100-fold excess ofnon-labeled HER2, the biotinylated ErbB2/Fc was captured on aneutravidin coated solid phase. Bound phage was specifically eluted withtrypsin (1 mg/ml).

In a fifth selection, phage libraries were incubated withHerceptin®-captured ErbB2/Fc. After extensive washing, bound phage wasspecifically eluted with trypsin (1 mg/ml)

In a sixth selection, soluble biotinylated ErbB2/Fc chimera wasincubated with the phage libraries. After extensive washing, thebiotinylated ErbB2/Fc was captured on a neutravidin coated solid phase.Bound phage was specifically eluted with Omnitarg-Fab.

In a seventh selection, phage libraries were incubated withHerceptin®-captured ErbB2/Fc. After extensive washing, bound phage wasspecifically eluted with Omnitarg-Fab.

In an eighth selection, phage libraries were incubated with biotinylatedextracellular domain of HER2 captured on a neutravidin coated solidphase. After extensive washing, bound phage was specifically eluted withOmnitarg-Fab.

In a ninth selection, phage libraries were incubated with biotinylatedextracellular domain of HER2 captured on a neutravidin coated solidphase. After extensive washing, bound phage was specifically eluted withHerceptin®.

In all selections, enrichment was observed. The output from eachselection was recloned as a pool into an expression vector derived frompUC119 which contained the LacZ promoter, a resistance gene forampicillin or carbenicillin, a multicloning site and the gen3 leadersequence. In frame with the Nanobody® coding sequence, the vector codedfor a C-terminal c-myc tag and a (His)6 tag. Colonies were picked andgrown in 96 deep-well plates (1 ml volume) and induced by adding IPTGfor Nanobody expression. Periplasmic extracts (volume: ˜80 μl) wereprepared according to standard methods (see for example the prior artand applications filed by applicant cited herein).

Example 4 Detection and Isolation of HER2-Specific Heavy Chain AntibodyProducing B-Cells

PBMC were isolated from peripheral blood samples from llamas immunizedwith HER2-Fc or SKBR3 human tumor cells using Ficoll density gradientcentrifugation. These were then resuspended in cell culture medium andpartially depleted from monocytes by adherence to the surface of plastictissue culture T-flasks.

Next, non-adherent PBMC were collected from the flasks, washed with FACSbuffer (PBS/10% FCS) at 4° C. and resuspended in the same ice-coldbuffer. These were then stained using a combination of Alexa 488 labeledHER2-Fc (produced in-house, using Invitrogen (Paisley, UK) activatedAlexa 488 and HER2-Fc recombinant protein from R&D Systems (Minneapolis,Minn.)), phycoerythrin labeled mouse-anti-llama IgG2 and -3 monoclonalantibodies (produced in-house, using purified phycoerythrin fromCyanotech, (Kailua-Kona, Hi.) crosslinked using the sulfo-SMCCheterobifunctional linker from Pierce-Endogen (Rochford, Ill.) toin-house produced and purified monoclonal antibodies originallydescribed in Daley et al. (Clin. Diagn. Lab. Immunol. 2005, 12: 380)),Alexa 647 labeled mouse-anti-llama IgG1 monoclonal antibody (producedin-house), Alexa 647 labeled mouse-anti-llama monocyte and neutrophilantibody DH59B (purified antibody obtained from VMRD Inc. (Pullman,Wash.)) and dead cell specific dye TOPRO3 (Invitrogen, Paisley, UK). Insome experiments, in-house Alexa 647 labeled recombinant human IgG1 Fcfragment (R&D Systems, Minneapolis, Minn.) was added to the staincombination as well.

Stained samples were washed thoroughly using cold FACS buffer andanalyzed on a standard two-laser BD FACSAria cell sorter equipped withthe ACDU microtiter plate single-cell deposition option (BD Biosciences,Franklin Lakes, N.J.). During acquisition and analysis, a gate was seton lymphocytes based on their forward/side scatter profile, whichoverlaps considerably with monocytes in llama. Doublet events wereeliminated from acquisition and analysis by forward as well as sidescatter pulse processing, eliminating all events which might beoriginating from more than one cell. Dead cells, monocytes and B-cellsexpressing conventional antibody on their cell membrane were removedfrom further analysis by gating out all remaining events havingfluorescence over background (unstained PBMC) in the Alexa647/TOPRO3channel. In some experiments, Alexa 647 labeled recombinant Fe fragmentwas used to stain the PBMC additionally. In these experiments, B-cellsproducing antibody binding Fc were also rejected from analysis andsorting by similar Alexa 647 channel exclusion, so as to avoid isolationof B-cells binding the Fe region of the fusion protein. In thephycoerythrin channel, B-cells displaying heavy chain antibody on theircell membrane could be clearly differentiated from any other remaininglymphocyte-type cells, and another gate was set on this population.Lastly, antigen-binding heavy chain IgG expressing B-cells cells weredetected as a discrete high fluorescence intensity peak population inthe Alexa 488 channel histogram distinct from the main population beingno more fluorescent in this channel than when no Alexa 488 labeledantigen was added. Individual antigen binding B-cells were collected inseparate wells of 96-well PCR plates in the ACDU, using DiVa softwarepredefined stringent single-cell sorting criteria to avoid anydouble-cell droplet or adjacent-droplet double cell sorting. Typically,only 1-5% of heavy chain B-cells were found to bind antigen.

Example 5 Amplification and Cloning of HER2-Specific Heavy ChainAntibody Variable Regions

Individual B-cells expressing heavy chain antibodies binding HER2-Fc orthe HER2 region of the fusion protein specifically were sorted into96-well plates containing 40 μl of RT-PCR buffer (Superscript IIIOne-step RT-PCR kit, Invitrogen, Paisley, UK) per well, as described inExample 5, and stored at −80° C. For variable region gene sequencerecovery, plates were thawed at room temperature and a mix of NP-40(Roche Applied Sciences, Indianapolis, Ind.), gene specific 5′ and 3′primers and RT-PCR enzyme mix were added to a total volume of 50microliter per well by an automated liquid handler (Tecan, Mannedorf,Switzerland). After reverse transcription and first PCR amplification ina standard thermal cycler, a 2 microliter aliquot was removed from allwells and amplified in a nested PCR reaction using a proof-readingthermostable polymerase, or blend of polymerases containing at least oneproof-reading enzyme. The 5′ nested primer contains the nucleotidesequence required for directional TOPO cloning (Invitrogen, Paisley,UK). The 3′ primer is designed to allow for the in-frame fusion ofvariable region gene framework 4 to vector encoded detection (c-myc) andpurification (6His) peptide tags. Amplicons were detected fromindividual wells using ethidium bromide stained agarose gels and/or inmicrotiter plates via PicoGreen DNA binding fluorescent dye assay(Invitrogen, Paisley, UK). Typically, up to 60% of wells contained asingle and sharply defined amplification product, whereas control wellsin the same plate not having received any cells were completely devoidof amplification product.

The amplicons from nested PCR wells containing detectable product werethen ligated into an E. coli expression vector in a homogenous ligationreaction, by mixing an aliquot of unpurified PCR mix with atopoisomerase-activated expression vector (in-house developed IPTGinducible E. coli Nanobody expression vector, adapted to allowdirectional TOPO cloning by Invitrogen's custom services department).The ligation mixture was then pipetted onto electrocompetent E. colicells pre-aliquotted in a 96-well format electroporation chamber array(BTX Products of Harvard Apparatus, Holliston, Mass.), and cells weretransformed by electroporation using a BTX pulse generator.

Transformation mix was spread on selective agarose, multiple individualsubcolonies picked and grown in 96-well deep well plates containingliquid selective medium by a QP Expression colony picker/rearrayersystem (Genetix, New Milton, Hampshire, UK).

Periplasmic extracts (volume: ˜80 μl) were prepared according tostandard methods (see for example the prior art and applications filedby applicant cited herein).

Example 6 Anti-HER2Nanobodies Recognize Extracellular HER2 Domain

Periplasmic extracts of individual Nanobodies were screened for HER2specificity by ELISA on solid phase coated ErbB2/Fc chimera (R&DSystems, Minneapolis, Minn.). Detection of Nanobody fragments bound toimmobilized recombinant HER2 antigen was carried out using an in housemade mouse anti-myc antibody (2 mg/ml) detected with alkalinephosphatase-conjugated anti-mouse IgG (Sigma Aldrich, Bornem. Belgium).The signal was developed by adding PNPP substrate solution and detectedat a wavelength of 405 nm. FIG. 2 is illustrative of typical ELISAresults, showing a high hit rate of positive clones.

Sequences of different HER2 binding clones are depicted in Tables B-1,B-2 and B-3. Alignment of the different HER2 binding clones based onCDR3 similarity is depicted in Table C-1.

Example 7 Anti-HER2Nanobodies Recognize Cell Surface Exposed ReceptorEpitopes

To verify whether the Nanobodies are able to recognize cell surfaceexpressed HER2, binding to breast cancer tumor cell line SKBR3 wasassessed by flow cytometry.

Cell binding assays were carried out by initially incubating 200,000cells with Nanobody-containing periplasmic preparation obtained inExamples 3 and 5 or relevant controls. After incubation, the cells werewashed with FACS buffer. Cells were subsequently incubated successivelywith an in-house mouse anti-myc-tag monoclonal antibody andphycoerythrin labeled goat anti-mouse F(ab′)2 fragments (JacksonImmunoResearch, Suffolk, UK). To omit signals arising from dead cells, aTOPRO-3 (Invitrogen, Paisley, UK) staining was carried out. Cells werefinally analyzed on a BD FACSArray Bioanalyzer System (BD Biosciences,Franklin Lakes, N.J., US).

FIG. 3 depicts binding of several Nanobody constructs to SKBR3 cells asmeasured by flow cytometric analysis. It can be seen that the constructs2A1, 2A3, 2C3, 2C5, 2D3 and 2G4 show clearly discernable shifts influorescence intensity as compared to the fluorescence intensity forcells incubated only with FACS buffer in the absence of any constructbut with all appropriate detection agents as used for the detection ofNanobody constructs.

Example 8 Screening for Nanobodies that Compete with Herceptin® for HER2Binding

A competition ELISA was performed to screen for Nanobodies that are ableto inhibit the Herceptin® interaction with HER2. In this competitionELISA, the binding of 2 nM Herceptin® to SKBR3 vesicles was evaluated inthe presence of a 1/20 dilution of Nanobody containing periplasmicextract obtained in the second selection described in Example 3. FIG. 4shows an example of this competitive ELISA, identifying several clonesthat compete with binding of Herceptin® to HER2 expressed on SKBR3vesicles.

Periplasmic extracts obtained in the second and ninth selectiondescribed in Example 3 and periplasmic extracts obtained in Example 5,were also screened in a Herceptin®-competitive homogeneous cell-basedassay to evaluate the capacity of the expressed Nanobodies to blockHerceptin® binding to HER2. The FMAT 8200 HTS system (AppliedBiosystems, Foster City, Calif.) assay was performed as follows: SKBR3cells expressing HER2 were grown in tissue culture flasks, collected andwashed with screening buffer (PBS, 10% FCS) and resuspended in screeningbuffer at a concentration of 2.5×10⁵ cells/ml. Alexa 647-labeledHerceptin® was diluted to 62.5 ng/ml in screening buffer. Periplasmicextracts were diluted in screening buffer to obtain final dilutions of4, 10, 40, 100, 200 and 400. To initiate the competitive screen, 10 μllabeled Herceptin®, 10 μl periplasmic dilution and 20 μl of cells wereadded to each well of FMAT system 384-well plates (PE Biosystems, FosterCity, Calif.) The plates were scanned after 2 hours of incubation. Awell was considered positive if it had a count of over 50 events.Screening of the extracts in this Herceptin® competitive homogeneouscell-based assay identified several clones (SEQ ID NOs: 1926-1988) thatcan block the binding of Herceptin® to HER2 with more than 90% (FIG. 5).

Purified Nanobodies were tested for inhibition of binding ofAlexa647-labeled Herceptin® to HER2 expressed on SKBR3 cells. Serialdilutions of purified Nanobody (concentration range: 20 nM-10 pM) wereadded to SKBR3 cells together with 4×10⁻¹⁰ M Alexa647-labeled Herceptin®and incubated for 2 hours, after which plates were scanned. Herceptin®was included as reference (MoAb). Results are shown in FIG. 6.Dose-response curves were observed for all Nanobodies with IC₅₀-valuesranging from 40 pM to 200 pM.

Example 9 Screening for Nanobodies that Compete with Omnitarg-Fab forHER2 Binding

Periplasmic extracts obtained in the sixth and seventh selectiondescribed in Example 3, were screened in an Omnitarg-Fab (OT-Fab)competitive homogeneous cell-based assay to evaluate the capacity of theexpressed Nanobodies to block OT-Fab binding to HER2. The FMAT 8200 HTSsystem (Applied Biosystems, Foster City, Calif.) assay was performed asfollows: SKBR3 cells expressing HER2 were grown in tissue cultureflasks, collected and washed with screening buffer (PBS, 10% FCS) andresuspended in screening buffer at a concentration of 2.5×10⁵ cells/ml.Biotinylated OT-Fab was diluted in screening buffer to obtain a finalconcentration of 0.586 nM. The periplasmic extracts were diluted inscreening buffer to obtain final dilutions of 100. To initiate thecompetitive screen, 5 μl labeled OT-Fab, 10 μl periplasmic dilution, 5μl FMAT Blue dye-labeled streptavidin (100 ng/ml) and 20 μl of cellswere added to each well of FMAT system 384-well plates (PE Biosystems,Foster City, Calif.). The plates were scanned after 2 hours ofincubation. A well was considered positive if it had a count of over 50events. Screening of the extracts in this OT-Fab competitive homogeneouscell-based assay identified clones that can block the binding of OT-Fabto HER2 with more than >90% (FIG. 7). Sequence analysis showed that allclones that blocked binding of OT-Fab HER2 are identical and represent asingle Nanobody (SEQ ID NO: 1989).

Example 10 Screening of Kinetic Off-Rate Constants Via Surface PlasmonResonance (BIAcore)

RhErbB2-Fc was immobilized on a CM5 sensor chip surface docked inBiacore 3000. Approximately 3600RU of rhErbB2-Fc was immobilized.Experiments were performed at 25° C. Periplasmic extracts were diluted10-fold in running buffer (HBS-EP). The samples were injected for 1 minat a flow rate of 45 μl/min over the activated and reference surfaces.Those surfaces were regenerated with a 3 s pulse of glycine-HClpH1.5+0.1% P20. As an example, the off rate (k_(off)) of differentNanobodies is documented in Table C-2.

Example 11 Anti-HER2Nanobodies can Block SKBR3 Cell Proliferation

The growth inhibitory characteristics of isolated Nanobodies wereevaluated using the breast tumor cell line SKBR3. Briefly, SKBR3 cellswere detached using 0.25% (vol/vol) trypsin and suspended in Dulbecco'sModified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum(FCS), glutamine, and penicillin-streptomycin at a density of 1×10⁵cells/ml. Aliquots of 200 μl (2×10⁴ cells) were plated into 96-wellmicrodilution plates and allowed to adhere. After overnight adherence,cells were washed with serum-free medium and starved for 4 hours in 100μl serum-free medium. Then, 100 μl of 1% FCS containing medium alone ormedium containing Nanobody (final concentration of 50 nM) was added.After 2 days of incubation, cells were pulsed with 1 μCi [³H]-thymidineand incubated for an additional 24 h prior to freezing at −80° C. Cellswere subsequently thawed and embedded on glass fiber membranes using acell harvester (Perkin Elmer Life Sciences, Wellesley, Mass., USA).After several washings with water, filters were air-dried and countedusing a γ-counter (Perkin Elmer Life Sciences). Nanobody 2A5 inhibitedSKBR3 proliferation by about 18%. Up to 30% or more inhibition wasachieved with Nanobodies 2C3, 2D3, 2A4 and 5F7 (FIG. 8).

Example 12 Generation of Multivalent/Multispecific Nanobody Formats

To potentially increase the biological effect of Nanobody molecules,bivalent constructs were fused head-to-tail using a GGGGSGGGS linker.

Here we describe the construction and characterization of bivalentNanobodies consisting of two identical anti-HER2 molecules all separatedby a 9 (GS) amino acid linker peptide. DNA segments encoding Nanobodies2A4, 2A5, 2C3, 2D3, 5F7 were head-to-tail fused resulting in constructs2A4-9GS-2A4, 2A5-9GS-2A5, 2C₃₋₉GS-2C3, 2D3-9GS-2D3, 5F7-9GS-5F7.Sequences of these bivalent constructs are listed in Tabel B-4. AllNanobodies were expressed in E. coli and purified according to standardprotocols (see for example the prior art and applications filed byapplicant cited herein).

The different bivalent Nanobody formats were screened in aHerceptin®-competitive homogeneous cell-based assay to evaluate theircapacity to block Herceptin® binding to HER2 compared to theirmonovalent format. Briefly, 10 μl labeled. Herceptin® (62.5 ng/ml), 10μl Nanobody dilution and 20 μl of cells (5×10³ cells) were added to eachwell of FMAT system 384-well plates (PE Biosystems, Foster City,Calif.). The plates were scanned after 2 hours of incubation. FIG. 9shows that the bivalent constructs are more efficient in blocking thebinding of Herceptin® to HER2-expressing SKBR3 cells as compared totheir monovalent formats.

To test whether selected Nanobodies have potential as anticancer agentsin an animal model, a strategy to increase the serum half life ispreferred (as for example described in patent application WO 04/041865),since the serum half life of a mono- or bivalent Nanobody (approximately15 or 30 KDa, respectively) is not optimal for this therapeuticindication. Human serum albumin specific Nanobody ALB1 (SEQ ID NO:2266), cross reactive with mouse serum albumin, was chosen. Here wedescribe the construction of bispecific Nanobodies consisting ananti-HER2Nanobody and ALB1, all separated by a 9 (GS) amino acid linkerpeptide and resulting in constructs 2A4-9GS-ALB1, 2A5-9GS-ALB1,2C₃₋₉GS-ALB1, 2D3-9GS-ALB1 and 5F7-9GS-ALB1. Sequences of thesebispecific constructs are given in Table B-5.

To test whether the HER2-binding Nanobodies as disclosed herein aboveretain their biological activity in a more complicated molecular contextsuch as a bispecific format, Nanobody formats were screened in aHerceptin®-competitive homogeneous cell-based assay to evaluate theircapacity to block Herceptin® binding to HER2 compared to theirmonovalent and bivalent format. Based on the results shown in FIG. 9, itcan be concluded that fusion of a Nanobody with different antigenspecificity to a HER2-binding Nanobody does not affect the potency ofthe latter.

Example 13 Generation of Biparatopic Formats Combining aHerceptin®-Competing Nanobody with a Library of HER2 Binding Nanobodies

The structural requirement for multispecificity is to fuse two or morebinding domains together, with sufficient flexibility to allowsimultaneous binding to different target epitopes. The simplestbispecific is one that binds to two different and non-overlappingepitopes on the same target in such a way that simultaneous binding tothe target is possible. Robert et al (Int. J. Cancer 1995, 28; 62(3):283-90) have described the design of high avidity biparatopic antibodiesdirected against two different epitopes of the carcinoembryonic antigen.Binding of both arms simultaneously without a significant loss ofentropy will endow ‘biparatopic’ antibodies with increased avidity andhence, increased binding affinity to the target. As a result, higherpotency can be obtained as well as enhanced selectivity. In addition,careful selection of the epitopes targeted on the antigen by thebiparatopic antibody or fragment thereof, combined with rational designof linkers to allow maximal flexibility of the two binding domainswithin the biparatopic antibody, may for example result in the blockingof two or more critical interaction sites of the target, leading toimproved potency.

Using genetic fusion, one Herceptin®-competing Nanobody was combinedwith a repertoire of HER2-binding Nanobodies and this mini-repertoirewas screened for biparatopics with improved binding activity and tumorcell growth inhibitory characteristics compared to the monovalentHerceptin®-competing Nanobody.

13.1 Construction of an Expression Vector for Biparatopic Design

For the construction of biparatope Nanobodies, an expression vector wasadapted to contain the Herceptin®-competitive Nanobody 2D3 (which wasshown to block cell proliferation between 20-30% as monovalent format(see Example 11) and which strongly competes with Herceptin® for bindingto HER2-overexpressing SKBR3 cells) to which other Nanobodies withdifferent HER2-binding specificities can be fused, spaced by a linker(FIG. 10). For the design of this vector, a 35 GS linker was used butother linker lengths can also be used to allow flexibility between thetwo building blocks. The 2D3 Nanobody is placed at the C-terminal end ofthe construct to allow SfiI-BstEII cloning of a full selection output.Alternatively, the 2D3 Nanobody can also be placed at the N-terminal endof the construct to allow cloning of a full selection output at theC-terminal end.

13.2 Generation of a Biparatopic Library

A full selection output retrieved from a selection onHerceptin®-captured rhErbB2/Fc followed by trypsin elution (Example3.4), was unidirectionally cloned to the 2D3 Nanobody. Sequence analysisof a selected number of individual colonies derived from the selectionoutput showed a good diversity in the repertoire: 16 Nanobody familieswere identified in 72 sequences. The ligation mix was transformed intoE. coli cells and the transformation mix spread on selective agarose.Multiple individual subcolonies were picked and grown in 96-well deepwell plates containing liquid selective medium by a QP Expression colonypicker/rearrayer system (Genetix, New Milton, Hampshire, UK).Fourty-eight individual colonies were sequenced and analyzed. From 32annotated sequenced, eight different Nanobody families were identified.

Periplasmic extracts (volume: ˜80 μl) were prepared according tostandard methods (see for example the prior art and applications filedby applicant cited herein). The biparatopic Nanobodies were purifiedfrom the periplasmic extracts using PhyTip200⁺ columns (Phynexus, SanJose, Calif.) by a Tecan Eva Robotic system (Promega, Madison, US) andanalyzed for their effects on SKBR3 tumor cell proliferation.

13.3 Effect of biparatopic Nanobodies on SKBR3 Cell Proliferation

The growth inhibitory characteristics of Nanobodies purified fromperiplasmic extracts by PhyTip200⁺ were evaluated using the breast tumorcell line SKBR3. Briefly, SKBR3 cells were detached using 0.25%(vol/vol) trypsin and suspended in DMEM supplemented with 10% fetal calfserum (FCS), glutamine, and penicillin-streptomycin at a density of1×10⁵ cells/ml. Aliquots of 200 μl (2×10⁴ cells) were plated into96-well microdilution plates and allowed to adhere. After overnightadherence, cells were washed with serum-free medium and starved for 4hours in 100 μl serum-free medium. Then, 100 μl of 1% FCS containingmedium alone or 90 μl of 1% FCS containing medium with 10 μl PhyTip200⁺purified periplasmic extract or 50 nM Herceptin® was added. After 2 daysof incubation, cells were pulsed with 1 μCi [³H]-thymidine and incubatedfor an additional 24 h prior to freezing at −80° C. Cells weresubsequently thawed and embedded on glass fiber membranes using a cellharvester (Perkin Elmer Life Sciences, Wellesley, Mass., USA). Afterseveral washings with water, filters were air-dried and counted using aγ-counter (Perkin Elmer Life Sciences).

Herceptin® was able to inhibit cell proliferation of SKBR3 up to 50%.Different subclasses of biparatopic Nanobodies were identified: a groupof biparatopic Nanobodies revealed an inhibitory effect on the ErbB2overexpressing cell line SKBR3 to a lower extent than Herceptin®, asecond group of biparatopic Nanobodies increased cell proliferation anda third group of biparatopic Nanobodies was able to inhibit cellproliferation of SKBR3 cells to an equal or greater extent thanHerceptin®. FIG. 11 shows an example of this ‘single hit’ cellproliferation assay.

Example 14 Characterization of Biparatopic Nanobodies

The biparatopic molecules 28F6-35GS-2D3, 28G5-35GS-2D3, 29E9-35GS-2D3,30D10-35GS-2D3, 27A5-35GS-2D3, 31D1′-35GS-2D3, 30E10-35GS-2D3,27A3-35GS-2D3, 27B7-35GS-2D3, 27C₃₋₃₅GS-2D3, 27D1-35GS-2D3,27E4-35GS-2D3, 27E7-35GS-2D3, 27H3-35GS-2D3, 27H4-35GS-2D3,27H5-35GS-2D3 were expressed in E. coli as c-myc, His6-tagged proteinsand subsequently purified from the culture medium by immobilized metalaffinity chromatography (IMAC) and size exclusion chromatography (SEC).A control biparatopic Nanobody consisting of a dummy (i.e. not bindingto HER2) Nanobody genetically fused to the 2D3 Nanobody, spaced by a35GS linker was used as a control.

14.1 Biparatopic Nanobodies Display Improved Binding to HER2 as Comparedto the Monovalent Building Blocks

The off-rate of the biparatopic Nanobodies was determined by surfaceplasmon resonance on a Biacore 3000 instrument. In brief, rhErbB2-Fc wasimmobilized on a CM5 sensor chip surface docked in Biacore 3000.Approximately 3600RU of rhErb B2-Fc was immobilized. Experiments wereperformed at 25° C. Nanobody binding was assessed at variousconcentrations. The samples were injected for 1 min at a flow rate of 45μl/min over the activated and reference surfaces to allow for binding tochip-bound antigen. Next, binding buffer without Nanobody was sent overthe chip at the same flow rate to allow for dissociation of boundNanobody. After 10 min, remaining bound analyte was removed by injectingregeneration solution (Glycine/HCl pH1.5).

The monovalent 2D3 and biparatopic dummy-2D3 Nanobodies had similaroff-rates in the range of 1E-3 1/s, indicating that fusion of a Nanobodyto the N-terminal end of 2D3 does not interfere with binding of thelatter (FIG. 12). The off-rate of bivalent 2D3-35GS-2D3 is in the rangeof 1E-4 1/s, indicating simultaneous binding of the two Nanobodies.

The off-rate of the biparatopic constructs 2B7-35GS-2D3, 27C3-35GS-2D3and 27H5-35GS-2D3 are in the range of 1E-3 1/s (FIG. 13). Theseoff-rates and the binding responses indicate binding by the 2D3paratope, but lack of binding by the other paratope, either bynon-specificity for rhErb2 or an extremely much lower affinity forrhErb2 compared to 2D3 or by sterical hindrance of the epitope by the Fcpart or by the altered conformation after the immobilization procedureon the CM5 sensor chip.

Off-rates of the biparatopic constructs 2D3-35GS-2D3, 27D1-35GS-2D3,27A3-35GS-2D3, 27E7-35GS-2D3 are in the range of 1E-4 1/s (FIG. 14).These off-rates indicate simultaneous binding of the 2 paratopes.

14.2 Herceptin®-Competitive Behavior of Biparatopic Nanobodies

Biparatopic Nanobodies were screened in a Herceptin®-competitivehomogeneous cell-based assay to evaluate the capacity of the expressedNanobodies to block Herceptin® binding to HER2. The FMAT 8200 HTS system(Applied. Biosystems, Foster City, Calif.) was used as described inExample 8. Bivalent 2D3-35GS-2D3 Nanobody more efficiently blocksbinding of Herceptin® to HER2 as compared to monovalent 2D3 (FIGS. 15Aand B). Likewise, biparatopic Nanobodies 27H3-35GS-2D3 and 27D1-35GS-2D3block binding of Herceptin® to HER2 expressed on SKBR3 cells moreefficiently than monovalent 2D3. Nanobodies 27A3, 27A5 and 30D 10 haveno influence on the Herceptin®-competitive characteristic of Nanobody2D3 when fused to its N-terminal end, spaced by a 35GS linker (FIG.15B). Finally, Nanobodies 27B7, 27C3, 27H5 and the dummy Nanobody havean inhibitory effect on the Herceptin®-competitive potential of 2D3(FIG. 15C).

14.3 Competitive Binding of Biparatopic Nanobodies with Omnitarg-Fab toHER2.

Biparatopic Nanobodies were screened in an Omnitarg-Fab competitivehomogeneous cell-based assay to evaluate the capacity of the expressedNanobodies to block Omnitarg-Fab binding to HER2. The FMAT 8200 HTSsystem (Applied Biosystems, Foster City, Calif.) was used as describedin Example 9. Biparatopic Nanobodies 2D3-35GS-2D3, 27H3-35GS-2D3,27D1-35GS-2D3, 27A3-35GS-2D3, 27A5-35GS-2D3 and 30D10-35GS-2D3 did notefficiently block the binding of biotinylated Omnitarg Fab (FIG. 16).Non-labeled Omnitarg-Fab inhibited binding of biotinylated Omnitarg-Fabin a dose-dependent manner.

Example 15 Biparatopic Nanobodies Comprising a Herceptin-Competitive anda HER2-Binding Nanobodies Inhibit SKBR3 Cell Proliferation

The growth inhibitory characteristics of biparatopic Nanobodies wereevaluated using the breast tumor cell line SKBR3. Briefly, SKBR3 cellswere detached using 0.25% (vol/vol) trypsin and suspended in DMEMsupplemented with 10% fetal calf serum (FCS), glutamine, andpenicillin-streptomycin at a density of 1×10⁵ cells/ml. Aliquots of 200μl (2×10⁴ cells) were plated into 96-well microdilution plates andallowed to adhere. After overnight adherence, cells were washed withserum-free medium and starved for 4 hours in 100 μl serum-free medium.Then, 100 μl of 1% FCS containing medium alone or 90 μl of 1% FCScontaining medium with serial dilutions of IMAC/SEC purified biparatopicNanobodies, monovalent 2D3 or 50 nM Herceptin® was added. After 2 daysof incubation, cells were pulsed with 1 μCi [³H]-thymidine and incubatedfor an additional 24 h prior to freezing at −80° C. Cells weresubsequently thawed and embedded on glass fiber membranes using a cellharvester (Perkin Elmer Life Sciences, Wellesley, Mass., USA). Afterseveral washings with water, filters were air-dried and counted using aγ-counter (Perkin Elmer Life Sciences).

Biparatopie Nanobodies are able to inhibit cell proliferation of SKBR3cells to an equal or greater extent than Herceptin®. FIG. 17 shows anexample of this cell proliferation assay.

Example 16 Biparatopic Nanobodies Comprising a Herceptin®-Competitiveand a HER2-Binding Nanobody Inhibit AKT Signal Transduction in SKBR3Breast Cancer Cells

Upon overexpression, HER2 may be activated by homodimerisation. HER2plays a major regulatory role in the signalling network involved in manycellular processes, including the p21Ras/Mitogen-Activated ProteinKinase (MAPK) and PI3K/AKT pathways. Treatment of HER2 overexpressingSKBR3 cells with Herceptin® results in reduction in HER2 phosphorylationwhich is linked to inhibition of AKT phosphorylation.

To assess the effect of biparatopic Nanobodies on the AKT pathway inSKBR3 cells, cells were plated in 2% serum containing medium in 24-wellculture plates. The next day, medium was refreshed and 50 nM of eitherbiparatopic Nanobody, Herceptin®, monovalent 2D3 Nanobody or mediumalone was added and incubated for 16 h. The reaction was stopped byaspirating the cell medium. Cells were lysed by addition of lysis buffer(20 mM NP40, 20 mM Tris-HCl pH8, 10% glycerol, 2 mM EDTA, 1 mM sodiumorthovanadate, complete protease inhibitor cocktail, 1% PBS). Proteinconcentration in the lysates was measured using BCA protein assay kit(Pierce) according to the manufacturer's indications. Equal amounts ofprotein were run on 10% polyacrylamide gels and electroblotted ontoInvitrolon PVDF membranes (Invitrogen, Paisley, UK). The presence ofposhorylated AKT was assessed by probing the blots with Phospho-AKT(Ser473) antibody (Cell Signaling, Danvers, Mass.) and total AKT wasdetected using AKT antibody (Cell Signaling). The blots were visualizedusing a chemiluminescent substrate (Perkin Elmer, Wellesley, Mass.,USA).

As shown in FIG. 18, biparatopic Nanobodies 27A5-35GS-2D3 and27A3-35GS-2D3 significantly block AKT activation in SKBR3 cells, whereasdummy-2D3 biparatopic and monovalent 2D3 Nanobody do not have a visibleeffect on AKT signalling.

Example 17 Construction of Biparatopic Nanobodies Combining Herceptin®-and Omnitarg Competitive Nanobodies

For the construction of biparatopics consisting of aHerceptin®-competitive and Omnitarg-competitive Nanobody, the expressionvector described in Example 13.1 was used. Herceptin®-competitiveNanobodies 2D3 and 5F7 were cloned either at the C-terminal orN-terminal end of Omnitarg-competitive Nanobody 47D5, spaced by a 35GSlinker. Biparatopic Nanobodies 2D3-35GS-47D5, 47D5-35GS-2D3,5F7-35GS-47D5 and 47D5-35GS-5F7 were expressed in E. coli as c-myc,His6-tagged proteins and subsequently purified from the culture mediumby immobilized metal affinity chromatography (IMAC) and size exclusionchromatography (SEC). Two control biparatopic Nanobody consisting of adummy Nanobody genetically fused to the 2D3 or 47D5 Nanobody, spaced bya 35GS linker were used as controls.

Example 18 Characterization of Biparatopic Formats Combining Herceptin®-and Omnitarg Competitive Nanobodies 18.1 Biacore Analysis

A kinetic analysis for 2D3, 5F7 and 47D5 was performed on Biacore todetermine the binding affinity to HER2. In addition, the influence of adummy Nanobody fused to the N-terminal end of 2D3 and 47D5 on thebinding characteristics of the latter to HER2, was analyzed. rhErbB2-Fcwas immobilized on a CM5 sensor chip surface docked in T100.Approximately 3600RU of rhErbB2-Fc was immobilized. Experiments wereperformed at 25° C. Different concentrations of Nanobody (100 nM-0.78nM) were made in running buffer (HBS-EP). The samples were injected for1 min at a flow rate of 45 μl/min over the activated and referencesurfaces.

In Table 2 an overview of k_(d)/k_(off), k_(a), and K_(d) values for theNanobodies is shown. Fusion of a Nanobody at the N-terminal end of theNanobodies 2D3 and 47D5 does not significantly alter the bindingcharacteristics of these Nanobodies to HER2.

The binding of the biparatopic 2D3-35GS-47D5 to HER2 was compared to thebinding of the monovalent building blocks 2D3 and 47D5. Hereto,approximately 90 RU of the respective Nanobodies were immobilized anddifferent concentrations (100-1000 nM) HER2-ECD was injected. As shownin Table C-4, the off-rate of the HER2-ECD from the 2D3-47D5 surface was25× lower than the off-rate on each of the 2D3 and 47D5 surfaces,indicating an avidity effect caused by binding of HER2-ECD on both the2D3 and 47D5 Nanobodies simultaneously (FIG. 19).

18.2 Biparatopic Nanobodies Combining Herceptin®- and OmnitargCompetitive Nanobodies Inhibit Heregulin-Mediated HER2-HER3 Signaling

After ligand-binding, the HER receptors become activated by receptordimerization between either two identical receptors (homodimerization)or different receptors of the same family (heterodimerization). Afterreceptor dimerization, activation of the intrinsic protein kinaseactivity and tyrosine autophosphorylation occurs, recruiting andphoshphorylating several intracellular substrates involving theRas-Raf-MAPK, the PI3K/Akt, and other signaling pathways that regulatemultiple biological processes including apoptosis and cellularproliferation. The mitogen-activated protein kinases (Erk1/Erk2) are oneof the key endpoints in signal transduction pathways that ultimatelytrigger cancer cells to divide.

The ability of the biparatopic Nanobodies combining Herceptin® andOmnitarg-competitive Nanobodies to inhibit heregulin (HRG) activation ofMAPK-Erk1/Erk2 was assessed in the following way. MCF7 cells(5×10⁴/well) were plated in serum-containing media in 24-well cultureplates. The next day, media were removed and fresh media containing 0.1serum were added to each well. The next day, prior to the assay, themedia were replaced with serum-free medium. Cells were then incubatedfor 30 min with 50 nM of biparatopic Nanobody 2D3-35GS-47D5,47D5-35GS-2D3, 5F7-35GS-47D5 or 47D5-35GS-5F7, monovalent 2D3, 5F7 or47D5, Omnitarg-Fab or Herceptin®. Cells were then treated with 0.2 nMHRG for 15 min. The reaction was stopped by aspirating the cell medium.Cells were lysed by addition of lysis buffer (20 mM NP40, 20 mM Tris-HClpH8, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, completeprotease inhibitor cocktail, 1% PBS). Protein concentration in thelysates was measured using BCA protein assay kit (Pierce) according tothe manufacturer's indications. Equal amounts of protein were run on 10%polyacrylamide gels and electroblotted onto Invitrolon PVDF membranes(Invitrogen, Paisley, UK). The presence of poshorylated Erk1/Erk2(p44/42 MAPK) was assessed by probing the blots with phosphor-p44/42MAPK (Thr202/Tyr204) antibody (Cell Signaling, Danvers, Mass.) and totalMAPK was detected using p44/42 MAP kinase (137F5) rabbit mAb (CellSignaling). The blots were visualized using a chemiluminescent substrate(Perkin Elmer, Wellesley, Mass., USA).

As shown in FIG. 20, biparatopic Nanobodies 2D3-35GS-47D5 and5F7-35GS-47D5 significantly block HRG-mediated activation of MAPK to agreater extent than Omnitarg-Fab and Herceptin®. Surprisingly, when theOmnitarg-competing Nanobody 47D5 comprised the N-terminal Nanobody inthe biparatopic constructs, i.e 47D5-35GS-2D3 and 47D5-35GS-5F7, nosignificant reduction in MAPK activation could be observed. MonovalentNanobodies 2D3, 5F7 and 47D5 could not block HRG-mediated MAPKactivation in MCF-7 cells.

These data suggest that the position of the Nanobodies within thebiparatopic Nanobody greatly influences the potency of the molecule. Inaddition, the length of the linker used to genetically fuse 2 Nanobodiesbiparatopic may be critically important to provide maximal flexibilitybetween the 2 Nanobodies to allow tight binding to their respectivebinding epitope on HER2.

Biparatopic Nanobodies 2D3-35GS-47D5 and 5F7-35GS-47D5 were also shownto inhibit heregulin (HRG)-dependent Akt activation (FIG. 21).Activation of the PI3K signal transduction pathway is important for cellsurvival. Complexes formed between HER2 and either HER3 or EGFR caninitiate these pathways in response to HRG. Incubation of MCF7 breastcancer cells with biparatopic Nanobodies 2D3-35GS-47D5 or 5F7-35GS-47D5inhibited HRG-mediated Akt activation to a greater extent thanOmnitarg-Fab or Herceptin®. These data suggest that the biparatopicNanobodies 2D3-35GS-47D5 or 5F7-35GS-47D5 may inhibit HER2ligand-activation of PI3 kinase and that this inhibition may lead toapoptosis.

Example 19 In-Silica Design of Optimal Linker Lengths in BiparatopicNanobody Formats

In-silico design of optimal linker lengths for a biparatopic Nanobodyformat may for example be performed as follows. The 3-dimensional (3D)coordinates of the binding mode of each individual Nanobody to itsrespective epitope on the target are determined, for example from:

-   -   a. a structure of the Nanobody-target complex determined by        X-ray experiments or NMR experiments.    -   b. a docking model of each Nanobody binding on their respective        epitope on the target. Also a number of potential binding modes        of each Nanobody to the target derived from docking studies can        be used. Docking can be done by e.g ZDock (Chen and Weng 2002,        Proteins 47(3): 281-294; Chen and Weng 2003, Proteins 51(3):        397-408; Chen et al. 2003, Proteins 52(1): 80-87) and refined by        RDock (Li et al. 2003, Proteins 53(3): 693-707) or by other        methods (Fernandez-Recio et al. 2003, Proteins 52(1): 113-117).    -   c. Binding mode of each Nanobody can be extracted from the same        structure or from separate complexes. In the latter case, the        binding modes of each Nanobody on a different epitope on the        same target can be deduced by structural superposition of the        different complexes.

A linker with a given sequence and thus of given length can be modelledbetween the 2 Nanobodies in different ways:

-   -   a. By homology modelling (Safi, and Blundell J. 1993, Mol. Biol.        234: 779-815)        -   i. The sequence of a construct Nanobody1-linker-Nanobody2 or            Nanobody2-linker-Nanobody 1 is drawn and stored in a            readable sequence format (e.g. Fasta)        -   ii. The 3-dimensional coordinates of the biparatopic            construct is built by homology modelling by using the            3-dimensional coordinates (from X-ray, NMR or docking            experiments) of the individual binding modes of the            Nanobodies as a template.    -   b. By de-novo design. A linker between the 2 Nanobodies binding        on a different epitope on the same target can be build by        de-novo design (Hu, et al. 2007, Proc. Natl. Acad. Sci. USA        104(45): 17668-17673).    -   c. Several conformations of the linker are sampled and the        lowest state energy conformations (1 or more) can be considered.

As a non-limiting example, the above was performed for a biparatopicconstruct comprising two Nanobodies. The modelling is shown in FIGS. 22Aand 22B, which show a model of Nanobody 2D3 (blue) linked to anotherNanobody (cyan) docked on HER-2 (red). Both figures show that we candock a Nanobody to a target and predict its binding mode to the target.When doing this for several. Nanobodies binding on non-overlappingepitopes on the same target, we can design a linker between theNanobodies to create a multivalent Nanobody construct.

The 3-dimensional coordinates of the in-silico generated linker in thebiparatopic construct are evaluated on at least one of the followingcriteria:

-   -   a. Internal energy strain of the linker; possibly compared to a        set of generated linkers of the same sequence in the free state.        At least one but preferentially several energy terms are used        (e.g. Van der Waals energy, electrostatics energy, dihedral        angle deformation energy, etc.). To calculate the energy values        an atom-based force-field (e.g. CHARMM (Brooks et al. 1983, J.        Comp. Chem. 4: 187-217)) or other means of calculating potential        energy (e.g. potentials of mean force (Muegge and Martin        1999, J. Med. Chem. 42: 791)) can be used.    -   b. Internal energy strain on at least one of the residues        (amino-acids) of the linker. For three biparatopic constructs        5F7-35GS-47D5, 47D5-35GS-5F7 and 47D5-40GS-5F7 energy penalty        values were calculated for each residue in the linker as well as        for 10 residues of each Nanobody connected to the linker. Energy        values are shown in FIGS. 22, 23 and 24.    -   c. The root-mean square deviation (RMSD) between the        3-dimensional coordinates of the 2 Nanobodies in the biparatopic        construct and the 2 Nanobodies in their non-linked (monovalent)        binding mode. The higher this value the less likely this linker        is appropriate. FIG. 25 shows the backbone RMSD (Å²) between the        5F7-linker-47D5 constructs (built by homology modelling) with        the individual Nanobodies 5F7 and 47D5 in their unlinked binding        mode. The linker length varies from 5 to 35. FIG. 25A shows that        the RMSD-value is at a minimum value with linker lengths larger        or equal to 15 residues. When shorter linkers are used (e.g.        linker length=5, 10) we see an increased RMSD indicating that        the Nanobodies in the bivalent construct are deviating from        their monovalent binding mode. These in-silico experiments        suggest that biparatopic constructs with linker lengths lower        than 15 residues will have a significant deviation from the        optimal binding mode of the individual Nanobodies to the target.        In FIG. 25B a ribbon view is shown of the 5F7-linker-47D5        biparatopic construct for 2 linker lengths. The binding mode of        the individual Nanobodies is shown in blue; the biparatopic        constructs are in red. When a 35GS linker is used between the 2        Nanobodies and a very limited deviation from the individual        binding modes is observed. However, when a 5GS linker is used,        both Nanobodies in the biparatopic construct significantly        deviate from their optimal binding mode.    -   d. Scores from scoring functions in homology modelling protocols        which are derived based on a combination of experimental data        and in-silico results (SalI & Overington, Protein Science        3(9):1582-1596, 1994).

As can be seen from the above results, the linker in this specificexample should preferably be at least 15 amino acids in length, withlinkers of between 20 and 40 amino acid residues, such as about 25, 30or 35 amino acid residues, being particularly suited.

Also, constructs with different potentially suitable linker lengths (asdetermined by the above in silico analysis) may be prepared and testedfor affinity/avidity, specificity, or potency using suitable bindingassays or in vitro or in vivo potency assays, for example thosementioned in the present specification. In this way, optimal linkerlength may be determined, confirmed or verified.

Example 20 Construction of Multiparatopic Nanobodies for BroaderBiological Activity

Simultaneous binding of 2 adjacent, non-overlapping epitopes by botharms of a biparatopic Nanobody without significant loss of entropyendows biparatopic Nanobodies with increased binding affinity to thetarget and as a result, higher potency can be obtained. For personsskilled in the art, it is evident that the engineering of Nanobodyfragments to obtain an increased potency or broader activity is notlimited to the construction of biparatopic Nanobody fragments.Engineering of triparatopic and even tetratopic Nanobodies with carefulselection of the epitopes targeted on the antigen, combined withselction of linkers to allow maximal flexibility of the binding domainswithin the multiparatopic antibody, may for example result in theblocking of several critical interaction sites of the target, leading toimproved potency and even an unparalleled biological activity.

Tables

TABLE B-1 Preferred Nanobodies against HER2 obtained as described inExample 3 ; PRT < Name , SEQ ID NO: # (protein) Amino acid sequence< 13D11 , SEQ ID NO: 1926 ; PRT; -> EVQLVESGGGLVHPGGSLRLSCVGSGFSLDDYGMTWVRRAPGKGLEWVSSINWSGTHTDYADSVKGRF TISRDNAKNTLFLQMNSLNPEDTAVYYCGQGWKIVPTNPRGHGTQVTVSS < 2B4 , SEQ ID NO: 1927 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVGSGFSLDDYAM TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKI RPTIPMGHGTQVTVSS < 2G2 , SEQ ID NO:1928 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYTDPVKGRF TISRDNAKNTLFLQMNNLTPEDTAVYYCNRGWKIVPTDLGGHGTQVTVSS < 13D2 , SEQ ID NO: 1929 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNNLRSEDTAVYSCNQGWKI VPTDRGGHGTQVTVSS < 2D5 , SEQ ID NO:1930 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF TISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRGGHGTQVTVSS < 2F4 , SEQ ID NO: 1931 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKI VPTDRRGHGTQVTVSS < 2C3 , SEQ ID NO:1932 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF TISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRTGHGTQVTVSS < 17E3 , SEQ ID NO: 1933 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTM GWYRQAPGKQRDLVASIDSSGGTNYADSVKGRFTISRDNAKNTVYLEMNSLTPEDTAVYYCNQGWKIV PTDRTGHGTQVTVSS < 17H3 , SEQ ID NO:1934 ; PRT; -> EVQLMESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF TISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKIVPTDRGGHGTQVTVSS < 17D2 , SEQ ID NO: 1935 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCNQGWKI VPTDRGSHGTQVTVSS < 2F1 , SEQ ID NO:1936 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKELEWISSINWSGTHTDYADSVKGRF TISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIVPMDRRGHGTQVTVSS < 2E2 , SEQ ID NO: 1937 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKI IPTDRRGHGTQVTVSS < 2C2 , SEQ ID NO:1938 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF TISRDNARNTLFLQMNSLTPEDTAIYYCNQGWKILPTDRRGHGTQVTVSS < 2E3 , SEQ ID NO: 1939 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKI LPTNRGSHGTQVTVSS < 13B10 , SEQ ID NO:1940 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGFEWVSSINWSGTHTDYADSVKGRF TISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKILPTNRGSHGTQVTVSS < 2D1 , SEQ ID NO: 1941 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNRGWKI LPTNRGSHGTQVTVSS < 2H3 , SEQ ID NO:1942 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF TISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS < 2H1 , SEQ ID NO: 1943 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM TWVRQAPGKGLEWVSSINWSGTHTDYADSVRGRFVISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKI IPTDRRGHGTQVTVSS < 2C1 , SEQ ID NO:1944 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF TISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS < 15C5 , SEQ ID NO: 1945 ; PRT; ->EVQLVESGGGLVQPGGSLKLSCVASGFSLDDYGM TWVRQAPGKGLEWVSSINWNVTHTDYAYSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKI IPTDRRGHGTQVTVSS < 2B3 , SEQ ID NO:1946 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDCADSVKGRF TISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS < 29H2 , SEQ ID NO: 1947 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNNLTPEDTAVYYCNQGWKI IPTDRRGHGTQVTVSS < 17E4 , SEQ ID NO:1948 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF VISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKIIPTDRRGHGTQVTVSS < 17A2 , SEQ ID NO: 1949 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAM TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYCNKGWKV WPTDRGTHGTQVTVSS < 15D1 , SEQ ID NO:1950 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF TISRDNAKNTLFLQMNSLNPEDTAVYYCNQGWKVWPTDRGTHGTQVTVSS < 17B8 , SEQ ID NO: 1951 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKI LPAERRGHGTQVTVSS < 15C11 , SEQ ID NO:1952 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF TISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKILPAERRGHGTPVTVSS < 15G8 , SEQ ID NO: 1953 ; PRT; ->EVQLVESGGGLVQPGGSLKLSCVASGFSLDDYGM TWVRQAPGKGLEWVSSINWNGTHTDYAYSVKGRFTISRDNAKNTLFLQMNSLTPENTAVYYCNQGWKI LPAERRGHGTQVTVSS < 17H4 , SEQ ID NO:1954 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASGFSLINYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF TISRDNAKNTLFLHMNNLSPEDTAVYYCGQGWKIHPADRGGHGTQVTVSS < 27G8 , SEQ ID NO: 1955 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDYGM TWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYCNQGWKI LPAERRGHGTQVTVSS < 38C6 , SEQ ID NO:1956 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCVGSGFSLDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF TISRDNAKNTLFLQMNSLSPEDTAVYYCNQGWKIRPTIPMGHGTQVTVSS < 2A4 , SEQ ID NO: 1957 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAM SWVRQAPGKGLEWVSAINWSGSHRNYADSVKGRFTISRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQS TTKNQGYWGQGTQVTVSS < 15G7 , SEQ IDNO: 1958 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSAINWSGTHRNYADSVKGRF TISRDNNKKTVYLQMNSLKSEDTAVYYCATGWQSTTKNQGYWGQGTQVTVSS < 15B7 , SEQ ID NO: 1959 ; PRT; ->EVQLVESGGGLVQPGGSLKLSCAASGFIFDDYAM SWVRQAPGKGLEWVSAINWSGSHRNYADSVKGRFTISRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQS TTKSQGYWGQGTQVTVSS < 5G4 , SEQ ID NO:1960 ; PRT; -> EVQLVESGGGLVQPGGSLTLSCAGSGFIFDDYAMSWVRQAPGKGLEWVSSINWSGSHRNYADSVKGRF TISRDNAKKTLYLQMNSLKSEDTAVYYCATGWQSTTKNQNYWGQGTQVTVSS < 13B2 , SEQ ID NO: 1961 ; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM SWVRQAPGKGLEWISSINWSGTHKDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRD AGTTWFEKSGSAGQGTQVTVSS < 2E5 , SEQ IDNO: 1962 ; PRT; -> EVQLVESGGSLVQPGESLRLSCAASGFTFDDYAMSWVRQAPGKGLEWISSINWSGTHTDYADSVKGRF TISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 15G1 , SEQ ID NO: 1963 ; PRT; ->EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAM SWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRD AGTTWFEKSGSAGQGTQVTVSS < 27B1 , SEQID NO: 1964 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWISSINWSGTHTDYADSVKGRF TISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 17E7 , SEQ ID NO: 1965 ; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM SWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRD AGTTWFEKSGSAGQGTQVTVSS < 17D8 , SEQID NO: 1966 ; PRT; -> EVQLVESGGSLVPPGGSLRLSCAVSGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF TISRNNANNMLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 5F8 , SEQ ID NO: 1967 ; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAL SWVRQAPGKGLEWISSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYCAKNWRD AGTTWFEKSGSAGQGTQVTVSS < 2D4 , SEQ IDNO: 1968 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRF TISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGPGTQVTVSS < 13D8 , SEQ ID NO: 1969 ; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM TWVRQASGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS < 17G8 , SEQID NO: 1970 ; PRT; -> EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSSINWSGTHTGYTDSVKGRF TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS < 2H4 , SEQ ID NO: 1971 ; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM TWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS < 2F3 , SEQ IDNO: 1972 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTGSVKGRF TISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGPGTQVTVSS < 2F5 , SEQ ID NO: 1973 ; PRT; ->EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAM SWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS < 30E10 , SEQID NO: 1974 ; PRT; -> KVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS < 29H1 , SEQ ID NO: 1975 ; PRT; ->EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYAM SWVRQAPGKGLEWVSSINWSGTHTGYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS < 17E2 , SEQID NO: 1976 ; PRT; -> EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS < 2B1 , SEQ ID NO: 1977 ; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM TWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGD AGTTWFEKSGSAGPGTQVTVSS < 2A5 , SEQ IDNO: 1978 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCATSGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS < 13C12 , SEQ ID NO: 1979 ; PRT; ->EVQLVESGGSLVQPGGSLRLSCATSGFTFDDYAM TWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS < 17E10 , SEQID NO: 1980 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDCTDSVKGRF TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS < 27D4 , SEQ ID NO: 1981 ; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM TWVRQASGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS < 15F9 , SEQID NO: 1982 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTGSVKGRF TISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS < 30H9 , SEQ ID NO: 1983 ; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM TWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGD AGTTWFEKSGSAGQGTQVTVSS < 39C1 , SEQID NO: 1984 ; PRT; -> EVQLVESGGSLVPPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRF TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS < 27G2 , SEQ ID NO: 1985 ; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAM TWVRQTPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYCAKNWGD AGTTWFEKSGSAGPGTQVTVSS < 2D3 , SEQ IDNO: 1986 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRF TISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 5F7 , SEQ ID NO: 1987 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGITFSINTM GWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAA QGTDYWGQGTQVTVSS < 118N121_A1_4_OK/ ,SEQ ID NO: 1988 ; PRT; -> EVQLVESGGGFVQTGGSPRLSCAASGRSFSEYAA 1-AWFRQSPGKERDLVAGIMWDGRSLFYADSVKGRF 127TISRDNAKNTLHLQMNSLKPEDTAVYYCAYHKTP YTTLELNRPHAFGSWGQGTQVTVSS < 47D5 ,SEQ ID NO: 1989 ; PRT; -> KVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSADSVKGRFT ISRVNAKDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSS < 14B11 , SEQ ID NO: 1990 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGSTFSSYGM GWFRQVPGKEREFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTARYYCGVETYGS GSSLMTEYDYWGQGTQVTVSS < 14B10 , SEQID NO: 1991 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAVNSRTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSIKGRFT ISRDNAKETVYLQMNSLKPDDTGVYYCAAETYGSGSSLMSEYDYWGQGTQVTVSS < 14B4 , SEQ ID NO: 1992 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAVSSRAFSSYGM GWFRQAPGKDREFVATINWSGVTAYADSIKGRFTISRDNAKETVYLQMNSLKPEDTGVYYCAAETYGS GSSLMSEYDYWGQGTQVTVSS < 14C11 , SEQID NO: 1993 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAVNSRTFSSYGMGWFRQAPGKEREFVATINWSGATAYADSIKGRFT ISRDNAKETVYLQMNSLKPDDTGVYYCAAETYGSGSSLMSEYDYWGQGTQVTVSS < 14B5 , SEQ ID NO: 1994 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAVSSRAFSSYGM GWFRQAPGKDREFVATINWSGVTAYADSIKGRFTISRDNAKETVYLQMNSLKPDDTGVYYCAAETFGS GSSLMSEYDYWGQGTQVTVSS < 14C6 , SEQ IDNO: 1995 ; PRT; -> EVQLVESGGGSVQAGGSLRLSCVASEGTFSSYGMGWFRQAPGKERAFVATINWSGVTAYADSVKGRFT ISRDNAKKTVYLQMNSLKPEDTAVYYCATDTYGSGSSLMNEYDYWGQGTQVTVSS < 14A4 , SEQ ID NO: 1996 ; PRT; ->EVQLVESGGGSVQAGSSLTLSCVASEGTFSSYGM GWFRQAPGKERAFVATINWSGVNAYADSVKGRFTISRDNAKKTAYLQMNSLKPEDTAVYYCAAETYGS GSSLMNEYDYWGQGTQVTVSS < 14B3 , SEQ IDNO: 1997 ; PRT; -> EVQLVESGGGLVQPGGSLTLSCVASEGTFSSYGMGWFRQAPGKERAFVATINWSGVNAYADSVKGRFT ISRDNAKKTAYLQMNSLKPEDTAVYYCAAETYGSGSSLMNEYDYWGQGTQVTVSS < 14C1 , SEQ ID NO: 1998 ; PRT; ->EVQLVESGGGSVQAGGSLRLSCAASGSTFSSYGM GWFRQAPGKERAFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCATETYGS GSSLMNEYDYWGQGTQVTVSS < 14A12 , SEQID NO: 1999 ; PRT; -> EVQLVKSGGGLVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSVKGRFT ISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLISEYDYWGHGTQVTVSS < 14A2 , SEQ ID NO: 2000 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASERTFSSYGM GWFRQAPGKEREFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGS GSSLISEYDYWGHGTQVTVSS < 14A1 , SEQ IDNO: 2001 ; PRT; -> EVQLVESGGGSVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSVKGRFT ISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLMSEYDYWGHGTQTVSS < 17C3 , SEQ ID NO: 2002 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAANGLTFRRYDM GWYRQAPGQQREWVAAISGAGDINYADSVKGRFTMARDNANHTVHLQMNSLKPEDTAVYYCNANWKML LGVENDYWGQGTQVTVSS < 46D3 , SEQ IDNO: 2003 ; PRT; -> KVQLVESGGGLVQAGGSLRLSCAASGRTFTEYSMGWFRQAPGKEREFVATISWNYGYTYYSDSVKGRF TVSRDIAENTVYLQMNTLKSEDTAVYYCAAKIGWLSIRGDEYEYWGQGTQVTVSS < 27H5 , SEQ ID NO: 2004 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYGI GWFRQASGKEREGVSCITSSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAALPFV CPSGSYSDYGDEYDYWGQGTQVTVSS < 17C2 ,SEQ ID NO: 2005 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYAMSWVRQAPGKGLEWVSAVDSGGGRTDYAHSVKGRF TISRDNAKNTLYLQMSSLKPEDTALYYCTKHVSDSDYTEYDYWGQGTQVTVSS < 17D11 , SEQ ID NO: 2006 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCTASGRTSSTSAM GWFRQAPGKEREFVATISRGGSATYYADSLKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAARRSS LYTSSNVFEYDYWGQGTQVTVSS < 15A6 , SEQID NO: 2007 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFT VSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSS < 17B6 , SEQ ID NO: 2008 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASRIPFSTRTM AWYRQAPGKQRDWVATIGTSGPPRYADSVKGRFTVSRDNAKNTVYLQMNSLKAEDTAVYYCWDVNADY WGQGTQVTVSS < 17C5 , SEQ ID NO: 2009; PRT; -> EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFT VSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTPVTVSS < 15E11 , SEQ ID NO: 2010 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASRIPFSSRTM AWYRQAPGKQRDWVATISARGMPAYEDSVKGRFTVSRDNDKNTLYLQMNSLKPEDTAVYYCRDVNADY WGQGTQVTVSS < 15C2 , SEQ ID NO: 2011; PRT; -> EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAQGKQRDWVATISSHGLPVYADSVKGRFT VSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSS < 2A3 , SEQ ID NO: 2012 ; PRT; ->EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTM AWYRQAPGKPRDWVATIRNGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLKPEDTATYLCRDVNGDIW GQGTQVTVSS < 27A5 , SEQ ID NO: 2013 ;PRT; -> EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQPPGNERDWVATIRSGAPVYADSVKGRFTV SRDNAKNTLYLQMNSLEPEDTATYYCWDVNGDIWGQGTPVTVSS < 2C5 , SEQ ID. NO: 2014 ; PRT; ->EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTM AWYRQTPGKSRDWVATIRSGTPVYADSVKGRFTVSRDNAKNTLYLRMNSLKSEDSATYTCRAVNADIW GQGTQVTVSS < 27G5 , SEQ ID NO: 2015 ;PRT; -> EVQLVESGGGLVQPGGSLRLSCVASRIPASIRTMAWYRQTPGNQRDWDATIGSSGTPAYADSVKGRFT VSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS < 13A9 , SEQ ID NO: 2016 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASRIPASIRTM AWYRQAPGKQRDWVATIGTGGTPAYADSFKGRFTVSRDNANHTVYLQMNSLKPEDTAVYYCRDVNGDY WGQGTQVTVSS < 29E9 , SEQ ID NO: 2017; PRT; -> EVQLVESGGGLVQPGGSLRLSCVASRIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFT VSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS < 15D8 , SEQ ID NO: 2018 ; PRT; ->EVQLVESGGGLVQPGGSLKLSCVASTIPASIRTM AWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDY WGQGTQVTVSS < 15G4 , SEQ ID NO: 2019; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGIPFRSRTMAWYRQAPGKTRDWVATIGTHGTPLYADSVKGRFT VSRDNAKNTLYLQMNSLKPEDTAVYYCWDVNGDYWGQGTQVTVSS < 15D12 , SEQ ID NO: 2020 ; PRT; ->EVQLVESGGGLVQAGESLRLSCATSGITFKRYVM GWYRQGPGKQRELVATVNDGGTTSYADSVKGRFAISRDNAKNTAYLQMNSLKAEDTAVYYCNAVWKLP RFVDNDYWGQGTQVTVSS < 15E12 , SEQ IDNO: 2021 ; PRT; -> EVQLMESGGGLVQAGGSLRLSCAANGLTFRRYDMGWYRQAPGQQREWVAAISGAGDINYADSVKGRFT MARDNANHTVHLQMNSLKPEDTAVYYCNANWKMLLGVENDYWGQGTQVTVSS < 13D7 , SEQ ID NO: 2022 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAANGLTFRRYDM GWYRQAPGQQREWVAAISGAGDINYADSVKGRFTMARDNANHTVHLQMNSLKPEDTAVYYCNANWKML LGVENDYWGQGTQVTVSS < 13A8 , SEQ IDNO: 2023 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGR FTISRDNAENTVYLQMNSLKPEDTAVYYCWDVNRDYWGQGTQVTVSS < 15A4 , SEQ ID NO: 2024 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRR TMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGRFTISRDNAKNTVYLQINSLKPEDTAVYYCWDVNR DYWGQGTQVTVSS < 17F7 , SEQ ID NO:2025 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGIAQSIRVMAWYRQPPGKQRDWVGTISSDGTANYADSVKGRFT ISRDNAKKTMYLQMNSLKPDDTAVYYCRDVNRDYWGQGTQVTVSS < 15C8 , SEQ ID NO: 2026 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGIAFRIRTM AWYRQAPGKQRDWVATSDSGGTTLYADSVKGRFTVSRDNAENTVYLQMNSLKPEDTAVYYCRDVNRDY WGQGTQVTVSS < 17A10 , SEQ ID NO: 2027; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASGIPSIRAIAWYRQAPGKQRDWVATSGTGYGATYDDSVKGRFTL SRDNAKNTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS < 27D3 , SEQ ID NO: 2028 ; PRT; ->EVQLMESGGGLVQPGGSLRLSCAASGLGIAFSRR TMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCWDVNR DYWGQGTQVTVSS < 13B12 , SEQ ID NO:2029 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGIAFRIRTMAWYRQAPGKQRDWVATIGSDGTTIYADSVKGRFT LSRHNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS < 15B2 , SEQ ID NO: 2030 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVVSGIPSSIRAM AWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLKPDDTAVYYCRDVNRD YWGQGTQVTVSS < 15B11 , SEQ ID NO:2031 ; PRT; -> EVQLVESGGGSVQAGGSLRLSCVVSGIPSSIRAMAWYRQAPGRQRDWVATIYSRSGGAVYADSVKGRF TISSDNAKNTIYLQMNSLKPDDTAVYYCRDVNRDYWGQGTQVTVSS < 13C9 , SEQ ID NO: 2032 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASGIPSIHAMA WYRQAPGKQRDWGATTYSRGGTTYNDSAKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCRDVNRDYW GQGTQVTVSS < 17D5 , SEQ ID NO: 2033 ;PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGIIGTIRTMAWYRQAPGKQRDWVASIGTRGAPVYADSVNGRFT ISRDGATNTVFLQMNNLKPEDTAVYYCRDVNRDYWGQGTQVTVSS < 27B5 , SEQ ID NO: 2034 ; PRT; ->EVQLVESGGGLVQAGGSLRLPCAASGIAFRIRTM AWYRQAPGKQRDWVATSDSGGTTLYADSVKGRFTVSRDNAENTVYLQMNSLKPEDTAVYYCRDVNRDY WGQGTQVTVSS < 27C7 , SEQ ID NO: 2035; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGIAFRIRTMAWYRQAPGKQRDWVATSDSGGTTLYADSVKGRFT VSRDNADNTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS < 13D4 , SEQ ID NO: 2036 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVVSGIPSSIRAM AWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLEPDDTAVYYCRDVNRE YWGQGTQVTVSS < 15G5 , SEQ ID NO: 2037; PRT; -> EVQLVESGGGLVQAGGSLRLSCVVSGIPSTIRAMAWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRF TISSDNAKKTIYLQMNSLKPDDTAVYYCRDVNREYWGQGTQVTVSS < 13C4 , SEQ ID NO: 2038 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVVSGIPSSIRAM AWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLKPDDTAVYYCRDVNRE YWGQGTQVTVSS < 46G1 , SEQ ID NO: 2039; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGRTFSDDAMGWFRQAPGKERECVASLYLNGDYPYYADSVKGRF TISRDNAKNAVILQMNNLKTEDTAVYYCAAKPGWVARDPSQYNYWGQGTQVTVSS < 46E4 , SEQ ID NO: 2040 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRAFKDDAV GWFRQAPGKERECVASMYLDGDYPYYADSVKGRFTISRDNAKNAVILQMNNLKTEDTAVYYCAAKPGW VARDPSEYNYWGQGTQVTVSS < 17B5 , SEQ IDNO: 2041 ; PRT; -> EVQLVESGGGLVQTGGSLRLSCAASGSTFRTDMMGWYRQAPGKQREFVASITKFGSTNYADSVKGRFT ISNDNAKDTVYLQMNSLKSEDTAVYYCRNFNRDLWGQGTQVTVSS < 15C9 , SEQ ID NO: 2042 ; PRT; ->EVQLVESGGGLVQAGGSLKLSCVNSGIPSTLRAM AWYRQAPGRQRDWVATSSNTGGTTYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCRDVNRDL WGQGTQVTVSS < 13D10 , SEQ ID NO: 2043; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASSVITLDSNAIGWFRQAPGKEREEVSCIASSDGSTYYAESVKGR FTISKDYTRNTVYLQVNSLKPEDTAVYHCATDANPNCGLNVWNSWGQGTQVTVSS < 17C6 , SEQ ID NO: 2044 ; PRT; ->EVQLVESGGGLVQAGGSLTLSCAASGSTSSLDIM AWYRQAPEKQRELVASVSGGGNSDYASSVKGRFTISGDTAKSTLYLQMNSLKPEDTAMYYCYGRDYYY MPFWGQGTQVTVSS < 15A2 , SEQ ID NO:2045 ; PRT; -> EVQLVESGGGLAQAGGSLSLSCAASGRFFSTRVMAWYRQTPGKQREFVASMRGSGSTNYADSARGRFA ISRDNAKNTVYLQMNSLKPEDTAVYYCRDINEDQWGQGTQVTVSS < 17A8 , SEQ ID NO: 2046 ; PRT; ->EVQLVESGGGLVQAGGSLSLSCAASGRFFSTRVM AWYRQTPGKQREFVASMRGSGSTNYADSVRGRFAISRDNAKNMVYLQMNTLKPEDTAVYYCRDINEDQ WGQGTQVTVSS < 15G10 , SEQ ID NO: 2047; PRT; -> EVQLVESGGGLVQAGGSLSLSCAASGRFFSTRVMAWYRQTPGKQREFVASMRGSGSTNYADSARGRFA ISRDNAKNTVYLQMNSLKPEDTAVYYCRDINEDQWGQGTQVTVSS < 27A3 , SEQ ID NO: 2048 ; PRT; ->EVQLVESGGGLVQAGGSLSLSCVASGRFFSTRVM AWYRQTPGKQREFVASMRGSGSTNYADSVRGRFAISRDNAKNTVYLQMNTLKPEDTAVYYCRDINEDQ WGQGTQVTVSS < 17H10 , SEQ ID NO: 2049; PRT; -> EVQLVESGGGLVQAGGSLSLSCSASGRFFSTRVMAWYRQTPGNQREFVATIHSSGSTIYADSVRGRFA ISRDNAKNTVYLQMRSLKPEDTAVYYCRDINADQWGQGTQVTVSS < 30D10 , SEQ ID NO: 2050 ; PRT; ->EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTM AWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDI WGQGSQVTVSS < 15H4 , SEQ ID NO: 2051; PRT; -> EVQLVESGGGLVQAGGSLTLSCAPSESTVSFNTVAWYRQAPGEQREWVATISRQGMSTYPDSVKGRFT ISRDNAKNTVYLQMNNLKPEDTAVYYCRDINHDIWGRGSQVTVSS < 17B7 , SEQ ID NO: 2052 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGIISSFRTM AWYRQAPGKQRDWVATIGSDGLANYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYFCRDINRDY WGQGTQVTVSS < 15D2 , SEQ ID NO: 2053; PRT; -> EVQLVESGGGLVQAGGSLRLSCVVSGVFGPIRAMAWYRQAPGKQRDWVATIGSSGHPVYTDSVKGRFT FSKDGAKNTVYLQMNSLKPEDTAVYYCRDINRDYWGQGTQVTVSS < 17G5 , SEQ ID NO: 2054 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGIGIAFSSR TMAWYRQAPGKQRDWVATIGSGGTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINR DYWGQGTQVTVSS < 15B6 , SEQ ID NO:2055 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGIIGSFRTMAWYRQAPGNQRDWVATIGSAGLASYADSVRGRFT LSRDNAKKTVYLQMNSLKPEDTAIYYCRDINGDYWGQGTQVTVSS < 27F2 , SEQ ID NO: 2056 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGIISSFRTL AWYRQAPGKQRDWVATISSAGGTAYADAVKGRFTISISRDNVEYTVDLQMDSLKPEDTAVYYCRDING DYWGQGTQVTVSS < 17F5 , SEQ ID NO:2057 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGR FTISRDNAKNTVYLQVNSLKPEDTAVYYCWDTNGDYWGQGTQVTVSS < 17B2 , SEQ ID NO: 2058 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAGSGFTFSNYAM TWVRQAPGKGLEWVSGVGGDGVGSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCTKDISTF GWGPFDYWGQGTQVTVSS < 27H4 , SEQ IDNO: 2059 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGWYRQAPGKQRDLVASIDASGGTNYADSVKGRFT ISRDNAKNTVYLEMNSLKPEDTGVYYCNGRWDIVGAIWWGQGTQVTVSS < 13A4 , SEQ ID NO: 2060 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTM GWYRQAPGKQRDLVASIDSSGGTNYADSVKGRFTISRDNAKNTVYLEMNSLKPEDTGVYYCNGRWDIV GAIWWGQGTQVTVSS < 2A1 , SEQ ID NO:2061 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCVASKITFRRYIMDWYRQAPGKQRELVASINSDGSTGYTDSVKGRFT ISRDNTKNTLDLQMNSLKPEDTAVYYCHGRWLEIGAEYWGQGTQVTVSS < 15E10 , SEQ ID NO: 2062 ; PRT; ->EVQLVESGGGLVQAGGSLKLSCVASGITFFRYTM GWYRQAPGKERELVAEISSADEPSFADAVKGRFTISRDNAKNTVVLQMNGLKPEDTAVYYCKGSWSYP GLTYWGKGTLVTVSS < 27E7 , SEQ ID NO:2063 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGITFRRYDMGWYRQFPGKERELVATILSEGDTNYVDPVKGRFT ISRDNAKNTVYLQMNDLKPEDTAVYYCNGVWRAIGRTYWGQGTQVTVSS < 47E5 , SEQ ID NO: 2064 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASASIFGFDSM GWYRQAPGNERILVAIISNGGTTSYRDSVKGRFTIARDNAKNTVSLQMNSLKPEDTAVYYCNLDRRSY NGRQYWGQGTQVTVSS < 2G4 , SEQ ID NO:2065 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGNIFSHNAMGWYRQAPGKQRELVTYITINGIANYVDSVKGRFT ISRDNTKNTMYLQMVSLKPEDTAVYYCNVGGREYSGVYYYREYWGQGTQVTVSS < 14D4 , SEQ ID NO: 2066 ; PRT; ->EVQLVESGGGLVQAGDSLRLSCAASGRALDTYVM GWFRQAPGDGREFVAHIFRSGITSYASSVKGRFTISRDNAKNTVYLQMASLKPEDTAAYYCAARPSDT TWSESSASWGQGTQVTVSS < 17A5 , SEQ IDNO: 2067 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFTEDDYSMSWVRQATGKGLEWVSGISWNGGSTNYADSVKGRF TISRDNVKNTLYLQMNSLKSEDTAVYYCAKDLGNSGRGPYTNWGQGTQVTVSS < 15D10 , SEQ ID NO: 2068 ; PRT; ->EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYRM YWVRQAPGKGLEWVSAIKPDGSITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDCGV PGFGWTFSSWGQGTQVTVSS < 13C2 , SEQ IDNO: 2069 ; PRT; -> EVQLVESGGGLVQAGGSLRLSCAASGSTFSINRMAWYRQSPGKQRELVAAVDNDDNTEYSDSVAGRFT ISRDNAKNAVHLQMNSLRLEDTAVYYCNAKQLPYLQNFWGQGTQVTVSS < 17G11 , SEQ ID NO: 2070 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGSTFSINRW GWYRQAPGKQRELVAAIDDGGNTEYSDFVNGRFTISRDNPETAVHLQMNSLKLEDTAVYYCNAKQLPY LQNFWGQGTQVTVSS < 17A3 , SEQ ID NO:2071 ; PRT; -> EVQLVESGGGLVQAGGSLSLSCAASATLHRFDNNWYRQAPGKQRELVATIAHDGSTNYANSVKGRFTI SRDNARDTLFLQMHALQPEDTAVYMCNLHRWGLNYWGQGTQVTVSS < 27B7 , SEQ ID NO: 2072 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAM SWVRQAPGKGLEWVSAISSGGGSITTYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTALYYCAKARS SSSYYDFGSWGQGTQVTVSS < 17A6 , SEQ IDNO: 2073 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISSGGGSITTYADSVKGR FTISTDNAKNTLYLQMSSLKPEDTALYYCAKARSSSSYYDFGSWGQGTQVTVSS < 17D7 , SEQ ID NO: 2074 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTLDYCAI GWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDRGS GTCYADFGSWGQGTQVTVSS < 46D4 , SEQ IDNO: 2075 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSSINWSGTHTDYAEDMKGRF TISRDNAKKTLYLQMNSLQSEDTAVYYCAKGWGPAVTSIPVATLGTQVTVSS < 27B3 , SEQ ID NO: 2076 ; PRT; ->EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTM AWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDI WGQGSQVTVSS < 27E5 , SEQ ID NO: 2077; PRT; -> EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFT ISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS < 27D6 , SEQ ID NO: 2078 ; PRT; ->EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTM AWYRQRPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDI WGQGSQVTVSS < 30D10 , SEQ ID NO: 2079; PRT; -> EVQLVESGGGLVQAGGSLTLSCTASETTVRIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFT ISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS < 47G11 , SEQ ID NO: 2080 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRIFYPMGW FRQAPGKEREFVAAIGSGDIITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASSRDYSR SRDPTSYDRWGQGTQVTVSS < 27C3 , SEQ IDNO: 2081 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYATSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRF TISRDNAKNTLYLQMNSLKPEDTAVYYCARPRGSSLYLLEYDYWGQGTQVTVSS

TABLE B-2 Preferred Nanobodies against HER2 obtained as described inExample 4 ; PRT < Name , SEQ ID NO: # (protein) Amino acid sequence< 11A101/1- , SEQ ID NO: 2082 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFNAMGW 120FRQAPGKEREFVAAISRSPGVTYYADSVKGRFTT SRDNAKNTVYLQMNDLKPEDTAVYYCAADFYLATLAHEYDYWGQGTQVTVSS < 11A22/1- , SEQ ID NO: 2083 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAM 122AWFRQAPGTEREFIAGIRWSDGSTYYADSVKGRF TISRDNAKNTVYLQMNSLKPEDTAVYYCAADFYVSTLAHEYDYWGQGTQVTVSS < 12D44/1- , SEQ ID NO: 2084 ; PRT; ->KVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAM 122AWFRQAPGTEREFIAGIRWSDGSTYYADSVKGRF TISRANAKNTVYLQMNGLKPEDTAVYYCAADFYVSTLAHEYDYWGQGTQVTVSS < 12E11/1- , SEQ ID NO: 2085 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAM 122AWFRQAPGKEREFVGGIRWSDGSTYYADSVKGRF TISRDNAKITVYLQMNSLKPEDTAVYYCAADFYVSTLAHEYDYWGQGTQVTVSS < 13G111/1- , SEQ ID NO: 2086 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAM 123GWFRQAPGKERAFVAAIRWSGGNTYYADSVKGRF TISRDNAKNTVYLQMNSLKPEDTAVYYCAADTFTLSTLSHEYDYWGQGTQVTVSS < 13E71/1- , SEQ ID NO: 2087 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASGRTFSNYAL 123AWFRQAPGKEREFVAAINWRSGGSTYYADSVKGR FTISRDNAKNTVYLQMNSLKPEDTAVYYCAADLIVATLPGEYDYWGQGTQVTVSS < 14H61/1- , SEQ ID NO: 2088 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRFAM 122GWFRQAPGKEREFVAAVRWSDDYTYYADSVKGRF TISRDNAKNTVYLQMNSLSPEDTAVYYCAADEILATLPHEYDYWGQGTQVTVSS < 22B12/1- , SEQ ID NO: 2089 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAM 124AWFRQAPGKEREFVAGINKSGGITHSADSVKGRF TISRDNAKNTVYLQMNSLKPEDTAVYYCAADAYTVIATLPHEYDYWGQGTQVTVSS < 14H71/1- , SEQ ID NO: 2090 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCEASGLTISSLTM 123AWFRQAPGKEREFVANIKWSGDRIVYADSVKGRF TISRDSAKNAVNLQMELVESDDTAVYYCAAKHSTVAGLTHEYDYWGQGTQVTVSS < 12D51/1- , SEQ ID NO: 2091 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGSAFSIKSM 120GWYRQAPGKQRELAAVIISSGTTTYADSVKGRFT ISRDSAKNTVYLQMDSLKPEDTAVYVCNAVYVSTWGNGYDYWGQGTQVTVSS < 11A111/1- , SEQ ID NO: 2092 ; PRT; ->EVQLVESGGGLVQAGGSLGLSCAAAGRTFSSSLM 126GWFRQAPGKEREFVAAITDNGGSTYYADSVKGRF TISRDNAKNSVYLQMNSLKPEDTAIYYCAARRSGYYSLSTSPHQYAYWGQGTQVTVSS < 13G71/1- , SEQ ID NO: 2093 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRAFSSYAM 124GWFRQAPGKERDFVAAITSSGSTNYADSVKGRFT ISRDNAKNTVYLQMNSLKPEDTAVYYCGARVNYAAYSRLEHDYHYWGQGTQTVSS < 13G74/1- , SEQ ID NO: 2094 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCATSGRTFSTYAS 125MGWFRQTPGKEREFVAAITSSGSTNYADSVKGRF TISRDNAKNTVYLQMNSLKPEDTAVYYCGARVNYAAYSRLEHDYHYWGQGTQVTVSS < 11A71A/1- , SEQ ID NO: 2095 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGNIDGIITM 116GWYRQRPGKPREWVGTINSGGDTNYAGSVKGRFT IARDDAKNTMYLQMNGMKPEDTAVYYCKMNRAGIYEYWGQGTQVTVSS < 22B101/1- , SEQ ID NO: 2096 ; PRT; ->EVQLVESGGGLVQTGGSLRLSCAASGPTFSDYAI 123GWFRQAPGKEREFVAAISSSGISTIYGDSVKGRF DISRDNAKNTVYLQMNRLKPEDTAVYYCAARLFMATPNQGQYYYWGQGTQVTVSS < 11B42/1- , SEQ ID NO: 2097 ; PRT; ->EVQLVESGGGLVQAGDSLRLSCAASGFTFSNHIM 123GWFRQAPGKERELIAHITWNGGSTYYADSVKGRF AISRDNALNTVYLQMNSLKPEDTAVYYCAARPSYSTNNVKSYRYWGQGTQVTVSS < 13E111/1- , SEQ ID NO: 2098 ; PRT; ->EVQLVESGGGLVQAGSSLRLSCALSGRTFSDYAI 124GWFRQAPGKEREFVAAISGWSGGTTNYADSVKGR FTISRDNGKNTVDLRMNSLKPEDTAVYYCAARPAVVHTRKESYPYWGQGTQVTVSS < 14H12/1- , SEQ ID NO: 2099 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCIASERTFSSAGV 125GWFRQAPGKERDFVAAISWNGVTIYYADSVKGRF TISRDNAKNTVYLQMNSLKPEDTAVYYCAARINYSVLTTTSSSYHYWGQGTQVTVSS < 13G101/1- , SEQ ID NO: 2100 ; PRT; ->EVQLVESGGGLVQPGDSLRLSCSASEGTLSRSRV 123AWFRQAPGKEREFVTVISGVGTSYADSVKGRFTI SRDDAKNTVYLQMNSLKAEDTAIYYCAADFRSTWLSSSGSSYTYWGQGTQVTVSS < 13G41/1- , SEQ ID NO: 2101 ; PRT; ->EVQLVESGGGLVQPGGSLTLSCVGSGRRFSADVM 121GWYRQAPGKQREFVASISSGSAINYADSVKGRFT VSRDNAQNTVYLQMNSLKIEDTGVYYCNARRIVNVEGAYRDYWGQGTQVTVSS < 22B910/1- , SEQ ID NO: 2102 ; PRT; ->EVQLVESGGGLVQPGGSLPLSCAASGSIFRMNDM 121GWYRQAPGKQRERVATLTSAGNTNYADSVKGRFT ISGDDARNTVYLQMNSLNPEDTAVYYCNAKVVVAVEGAKYDYWGQGTQVTVSS < 21A81/1- , SEQ ID NO: 2103 ; PRT; ->EVQLVESGGGLAQAGGSLRLSCAVFGRSRYGMAW 122FRRAPGKEREFVAGIAWNGASIGSADSVRGRFTI SRDNSENTVYFEMGSLKPEDTAVYYCAICRISWCAGAESDYGYWGQGTQVTVSS < 21A92/1- , SEQ ID NO: 2104 ; PRT; ->EVQLVESGGGQVQAGGSLRLSCTESGRAFNTRAM 127GWFRQAPEKEREFVAGITMSGFNTRYADSVKGRF TISRDNAKGTVYLQMSSLKPEDTAVYYCAADSITDRRSVAVAHTSYYYWGQGTQVTVSS < 22C712/1- , SEQ ID NO: 2105 ; PRT; ->EVQLVESGGGLVQAGGSLGLSCAASGRTFSNYAM 123GWFRQAPGKEREFVAGISWSGGHTFYADSVKGRF TISRDNTKNTVYLQMNSMRPEDTAVYYCAARLSSVAVASTRYDYWGQGTQVTVSS < 11A13/1- , SEQ ID NO: 2106 ; PRT; ->EVQLVESGGGLVQAGDSLRLSCVASGGTFGSYAM 125GWFRQAPGKEREFVATIDWSGDTAFYADSVKGRF TISRDIANDVVYLQMNSLEPEDTAVYYCARNRQSGVASENLRLYTYWGQGTQVTVSS < 13G93/1- , SEQ ID NO: 2107 ; PRT; ->EVQLVESGGGLAQAGDSLRLSCVDSGSSFSAYAM 123GWFRQAPGKEREFVAAVSWDGRNTYYADSVKGRF TISRDNAKNTLYLQTTSLRPEDTGVYYCAEDKQSGVSVNPKYAYWGQGTQVTVSS < 12C52/1- , SEQ ID NO: 2108 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAVSGGTFESDTM 118AWFRQAPGKEREFVARVSWIRTTYYSDSVKGRFT ISKDNAKNTVYLQMNSLKPEDTAVYYCAAQTLGRSLYDYWGQGTQVTVSS < 12C61/1- , SEQ ID NO: 2109 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSSNAM 126AWFRQAPGNERELVSAIGWSGASTYYIDSVEGRF TISRDNAKNTVYLQMNSLKPEDTAVYYCAASRYSGGVATARRSEYHYWGQGTQVTVSS < 21A61/1- , SEQ ID NO: 2110 ; PRT; ->EVQLVESGGGLVQAGDSLRLSCVASGDSFNTYTM 125GWFRQAPGKERFEVAAIRWSGGTTFYGDSVKGRF TISRDYAKNTWYLQMNTLKPEDTAAYYCAAVATYSRNVGSVRNYDYWGQGTQVTVSS < 11A121/1- , SEQ ID NO: 2111 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVVSEGTFSSYSM 126GWFRQAPGKDREFVSAITWNGTRTYYRDSVKGRF TISRDNAKNTVQLQMNSLKPEDTAVYYCAVSQPLNYYTYYDARRYDYWGQGTQVTVSS < 11A91/1- , SEQ ID NO: 2112 ; PRT; ->EAQLVESGGGLVQAGGSLRLSCTASGRTYSTTMG 124WFRQAPGKEREFVAAIRWSGGSAFYADSVKGRFT ISRDNAKNTVYLQMTSLMPEDTAVYYCADTPVYYQRYYDQNAYDYWGQGTQVTVSS < 13G72/1- , SEQ ID NO: 2113 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRAFSSYAM 118GWFRQAPGKERDFVAAITSSGSTNYADSVKGRFT ISRDNAKNTVYLQMNSLKPEDTAVYYCAAKYYSYYAYDYWGQGTQVTVSS < 13E81/1- , SEQ ID NO: 2114 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGGTFSVYHM 124AWFRQAPGKEREFVAAIRSSGGLFYALSVSGRFT ISRDNAKDTMYLQMNVLKPEDTAVYYCAASPVYYIDYSSQYKYGYWGQGTQVTVSS < 11B31/1- , SEQ ID NO: 2115 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGGAFGVYHM 124GWFRQAPGKEREFVAAIRSGGTTLYEDSVKGRFT ISRDNAKNTVYLRMNSLKPEDTAVYYCATQIYYRTNYYSQNAYDYWGQGTQVTVSS < 13G81/1- , SEQ ID NO: 2116 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGGTFGVYHM 124GWFRQAPGKEREFVAVIRSGGTTLYADSVKGRFT ISRDDAKNTVYLQMNSLKPEDTAVYLCAAQIYYRTNYYSQNNYDYWGQGTQVTVSS < 21A53/1- , SEQ ID NO: 2117 ; PRT; ->EVQLVESGGGLVQAGGSLELSCAASGGAFGVYHM 124GWFRQAPGKEREFVAAIRSGGTTLYEDSVKGRVT ISRDDAKNTVYLRMNSLKPEDTAVYYCAAQIYYRTNYYSQNVYDYWGQGTQVTVSS < 14H51/1- , SEQ ID NO: 2118 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGGTFGVYTM 124AWFRQAPGKEREFVAAIRSGATTLYEDSVKGRFT ISRDDAKNTVYLRMNSLKPEDTAVYYCAAQIYYRTNYYSQNEYDYWGQGTQVTVSS < 21A21/1- , SEQ ID NO: 2119 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGGTFGVYHM 124GWFRQAPGTEREFVAVIRSGGTTLYEDSVKGRFT ISRDNAKNTVYLRMNSLKPEDTAVYYCAAQIYYRTNYSSQSNYDYWGQGTQVTVSS < 21A111/1- , SEQ ID NO: 2120 ; PRT; ->EVQLVESGGGLVQAGGSLKLSCAVSGRTIVPYTM 124AWFRQAPGKEREFVAVTRSGGTTFYADSAKGRFT IARDDAKNTVYLQMNSLKPEDTAVYYCALATAYRTNYSSRDKYDYWGQGTQVTVSS < 22B1212/1- , SEQ ID NO: 2121 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAM 122SWVRQAPGKGLEWVSAINSGGGSTSYADSVKGRF TISRDNAKNTLYLQMNSLKPEDTAVYYCAKYLSFYSDYEVYDYWGQGTQVTVSS < 11A31/1- , SEQ ID NO: 2122 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGGTFSSGVM 120AWFRQSPGEEREFLALITRNGETKKTADSVKGRF TISRDNAKNGVSLQMDSLKAEDTAVYYCASDPTYGSGRWTYWGQGTQVTVSS < 13E51/1- , SEQ ID NO: 2123 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASRHTFSGYAM 128GWFRQAPGKEREFVAAIRWSGGITYYADSVKGRF TISSDNAKNTVYLQMNSLKPEDTALYYCARSVTYYSGSHAYTQEGGYARWGQGTQVTVSS < 12D121/1- , SEQ ID NO: 2124 ; PRT; ->EVQLVESGGGLVQTGGSLRLSCAASGRAFSTYGM 126GWFRQAPGKAREFVAAISRSGTGTYYAGSMKGRF TISRDDAKNTVYLQMNSLKPEDTAVYYCAARQPYASGSHYSSTQYTYWGQGTQVTVSS < 13F121/1- , SEQ ID NO: 2125 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRSFNDYTM 119GWFRQTPGKEREFVARVWWNGGSAYYADSVKGRF TISIDNAKNTVYLQMNNLTPEDTAVYYCAALYRGRSVYDDWGQGTQVTVSS < 13G121/1- , SEQ ID NO: 2126 ; PRT; ->EVQLVESGGGLVRAGTSLRLSCADSARTFSSAAM 127GWFRQAPGKEREFVSAISPIGSSKYYADSVKGRF TISRDNAKNTVYLQMDSLKPEDTAVYYCAASSYGSTYYSQGRAYYYDYWGQGTQVTVSS < 22B41/1- , SEQ ID NO: 2127 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCTVFGRTFSGDVI 124GWFRQAPGKEREFVAAISTSGGGTDSADSVKGRF TISKENAKNTVYLQMTILKPEDTAVYYCASSPYGPLYRSTHYYDYWGQGTQVTVSS < 12D71/1- , SEQ ID NO: 2128 ; PRT; ->EVQLVESGGGLVQAGGSLGLSCAASGRTVSTMGW 125FRQAPGKEREFVTAITWSGDSTNFADSVKGRFTI SRDSAKDTVYLQMNNLKPEDTAVYYCAATTYYSGSYISTLSTSYNYWGQGTQVTVSS < 13F42/1- , SEQ ID NO: 2129 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASGRTLSTTGV 111GWFRQAPGKGRESVATIFVGGTTYYSDSVKGRFT ISRDNAKNAVNLQMSNLKPEDTALHYCTIGSYRGQGTQVTVSS < 12C101/1- , SEQ ID NO: 2130 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASGRTLSTTGV 111GWFRQAPGKERESVATIFVGGTTYYSDSVKGRFT ISRDNARNAVNPQMNNLKPEDTAVYYCTIGSYRGQGTQVTVSS < 14H91/1- , SEQ ID NO: 2131 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRDVM 127GWFRQAPGKEREFVAAKTWSGASTYYADSVRGRF TISRDNAKNAVYLQMNSLKPEDTAVYYCAARDSSTLDSTYYVGGSYNYWGRGTQVTVSS < 13F41/1- , SEQ ID NO: 2132 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASGRTLSTTGV 111GWFRQAPGKERESVATIFVGGTTYYSDSVKGRFT ISRDNAKNAVNLQMSNLKPEDTALYYCTIGSYRGQGTQVTVSS < 14H21/1- , SEQ ID NO: 2133 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVRSGGYFGSYHI 125GWFRQAPGNEREFVAAITWNGASTSYADSVKGRF TISRSIAENTVYLQMNKVKPEDTAVYYCAARMYGSDWLPRPEDFDSWGQGTQVTVSS < 22B610/1- , SEQ ID NO: 2134 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGSIFSINAM 120GWYRPAPGKQRELVARITSTGSTNYADSVKGRFT ISRDNAKNTVYLQMNSLKPEDTAVYYCNADVSPSYGSRWYGWGQGTQVTVSS < 12C32/1- , SEQ ID NO: 2135 ; PRT; ->EMQLVESGGGLVQAGGSLRLSCATSERTFSTYTM 127AWFRQAPGKEREFVVAIKSSDNSTSYRDSVKGRF TISRDNAKSTMYLQMNSLKPEDTAVYYCAARREYSTIYTARYPGEYVYWGQGTQVTVSS < 12D61/1- , SEQ ID NO: 2136 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASRSIFSPNVV 116GWYRQAPGKQRELVAAVTSGGITNYADSVKGRFT ISRDNAKNTLYLQMNSLKAEDTAVYYCNARERGIYDSWGQGTQVTVSS < 13G31/1- , SEQ ID NO: 2137 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGGTFSRYKM 125GWFRQAPGKEREFVAASRWSGGIKYHADSVKGRF TISRDDAKNSIYLQMNTLKPEDTAVYYCAADDYLGGDNWYLGPYDSWGQGTQVTVSS < 22C65/1- , SEQ ID NO: 2138 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAVSGFLFDSYAM 124GWFRQAPGKEREFVAAIRWSGSATDYSDSVKGRF TISRDNAKNTVYLQMNSLIPEDTAVYYCAARKTYRSLTYYGEYDSWGQGTQVTVSS < 11A71/1- , SEQ ID NO: 2139 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASRSIRSVSVM 125GWYRLAPGNQRELVATITADGITNYADSVKGRFT VSRDNGRNTVYLQMNSLKPEDTAVYYCNVDRLLYYSSGYYQTSVDVWGQGTQVTVSS < 11B91/1- , SEQ ID NO: 2140 ; PRT; ->EVQLVESGGALVQPGGSLRLSCAASGSIRSINTM 125GWYRQAPGNQREFVAAVTEGGTTSYAASVKGRFT ISRDKAKNTVLLQMDSLKPEDTAVYYCNADRFLYYSAGRYDTGSDIWGQGTQVTVSS < 11A81/1- , SEQ ID NO: 2141 ; PRT; ->EVQLVESGGALVQPGGSLRLSCAASDSIRSINIM 125GWYRQAPGKQREFVAAVTEDGSINYAESVKGRFT ISRDKAKNALYLQMNSLKPEDMAVYYCNADRVLYYSDSRYYTGSNYWGQGTQVTVSS < 11B121/1- , SEQ ID NO: 2142 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGSSASINTM 127GWYRQAPGEQRELVAEITEGGIINYTDSVKGRFT ISRDNAKNTVYLEMNNLKPEDTAVYYCNADRALYRNYSDGRYYTGYDYWGQGTQVTVSS < 12D31/1- , SEQ ID NO: 2143 ; PRT; ->EVQLVESGGGEVQPGGSLRLSCAASRNIFDFNDM 115GWYRQGPGKEREFVALINVGGVAKYEDSVKGRFT ISRDNAENTVYLQMNNLKPEDMAVYYCNARILSRNYWGQGNQVTVSS < 11B51/1- , SEQ ID NO: 2144 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGGTFSGRGM 127GWFRQAPGKEREFVAAVSWSGGNTYYADSVKGRF TISRDNAKSTVYLQMDSLKPEDTAVYYCAASRRFYSGLYYYTDDAYEYWGQGTQVTVSS < 13G51/1- , SEQ ID NO: 2145 ; PRT; ->EVQLVESGGGLVQAGGSLSLSCAASGGTFNGRAV 127GWFPQAPGEEREFVTGISWSGGSTDYADSVKGRF TISRDNSKNTVSLQMNSLKPEDTAVYYCAASRRFYSGLVYYSVDAYENWGQGTQVTVSS < 13F82A/1- , SEQ ID NO: 2146 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAISGRTLSGRAM 130GWFRQAPGKEREFREFVAATSWSGGSKYVADSVT GRFTIFRDNAENTAYLQMNSLNPEDTAVYYCAVTKRYYSIKYYSTVEDYEYWGQGTQVTVSS < 13E101/1- , SEQ ID NO: 2147 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAVSGRTFNNDHM 128GWFRQAPGTERELVAATGRRGGPTYYADSVKGRF TISRDNAESTVYLQMNSLKAEDTAVYYCAANRYYCSTYGCLSTPRQYDYWGQGTQVTVSS < 22B85/1- , SEQ ID NO: 2148 ; PRT; ->EVQLVESGGGLVRPGGSLRLSCATSGSDIGINAM 120GWYRQAPGNQRELVATITGSTGTTYADSVKGRFA ISRDGAKNTVYLQMDSLKPEDTAVYYCNLRVYTGTYGGRNYWGQGTQVTVSS < 11B12/1- , SEQ ID NO: 2149 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRALINYAM 118GWFRQAPGKEREFVSAINWSGSHTDYGDSVKGRF AISRDNAKNTVYLQMHSLKPEDTAVYHCATGYSLPAFDSWGPGTQVTVSS < 13G61/1- , SEQ ID NO: 2150 ; PRT; ->EVQLVESGGGVVQAGGSLRLSCAPSGRTFSSYVM 118GWVRQAPGKAREFVAGITRNSGRTRYADSVKGRF TISRDNADNTVTLQMNSLKPEDTAVYYCAGGIDLYTFHYFGQGTQVTVSS < 14H41/1- , SEQ ID NO: 2151 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAPSGRTFSSYVM 118GWVRQAPGKAREFVAGITRNSIRTRYADSVKGRF TISRDNADNTVTLQMNSLKPEDTAVYYCAGGIDLYTFDYFGQGTQVTVSS < 11B81/1- , SEQ ID NO: 2152 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRPVNNYIM 126GWFRQALGQGREFVAAINRNGATAAYADSVKGRF TISRDNAEDLLYLQMNLLKPEDTAVYYCAANSDSGFDSYSVWAAYEYWGQGTQVTVSS < 11C11/1- , SEQ ID NO: 2153 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSAYAM 121GWFRQAPGKERESVATIRWTGGSSSTSYADSVKG RFTISKNTAENTVYLQMNSLKPEDTAVYYCAVLLTVWDTYKYWGQGTQVTVSS < 12D92/1- , SEQ ID NO: 2154 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTYNMAWF 123RQAPGKEREFVAAMNWSGGSTKYAESVKGRFTIS RANDNNPLYLQMNTLKPEDTAVYYCAATNRWYTGVYDLPSRYEYWGQGTQVTVSS < 13E61/1- , SEQ ID NO: 2155 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCTASGQTFNMGWF 123RQAPGKEREFVAAISWSQYNTKYADSVKGRFTIS RDNAINSLYLQMDTLKPEDTAVYYCAATNRWFSAVYDLPSRYTYWGQGTQVTVSS < 22B71/1- , SEQ ID NO: 2156 ; PRT; ->EVQLVESGGAFVQPGGSLRLSCAASGSDVWFNVM 114GWYRQGPGQQLELVASITYGGNINYGDPVKGRFS ISRDNALKTVYLQMNSLKPEDTAVYYCYADLPSRLWGQGTQVTVSS < 21A121/1- , SEQ ID NO: 2157 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCTASGRAFNMGWF 123RQAPGKEREFVAGVNWGGGSTKVADSVKERFTIS RDYDNSPVYLQMNTLKPEDTAVYYGAATSRWYSAVYDLPTRYDYWGQGTQVTVSS < 13F101/1- , SEQ ID NO: 2158 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCQLSGGTVSDLHM 124GWFRQAPGKEREFVGFTRWPSITYIAEHVKGRFT ISRDNAKNTVYLQMNSLEREDTAVYYCAADRSYSIDYRHPDSYSYWGQGTQVTVSS < 11A43/1- , SEQ ID NO: 2159 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGSIFRVNHM 123GWYRQAPGKQREFVAAITSDHITWYADAVKGRFT ISRDNAKNTVTLQMNSLRPEDTAVYYCAADPLLFYGVGSADVDYWGQGTQVTVSS < 12C81/1- , SEQ ID NO: 2160 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAGSGNIVRDNTM 117AWYRQAPGNQRDLVATINVGGGTYYAGPVKGRFT ISRDNAKNSVYLQMNSLKPEDTSVYYCNVISGLVQRDYWGQGTQVTVSS < 11B21/1- , SEQ ID NO: 2161 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSMYLM 124GWFRQAPGKEREFVSTINRRGGNTYYADSVKGRF TISRDNARNTVYLQMNSLKPEDTAVYYCAAGGHLLGYDVQWEPDYWGQGTQVTVSS < 11B71/1- , SEQ ID NO: 2162 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFERYAM 126GWFRQAPGKEREFVATISWSGGRDTVYADSVKGR FTISRDNAKNTVYLQMNSLKPEDTAVYYCAAHKRTYELGAHSTDFGSWGQGTQVTVSS < 12C121/1- , SEQ ID NO: 2163 ; PRT; ->EVQLVESGGDLVQPGESLRLSCAVSGVTVDYSGI 126GWFRQAPEKEREAVSCIESGDGTTTYVDSVKGRF TISRDNAKNAVYLQMNSLKPEDTGVYYCATAVFVDSGDFSVCRGVGYWGKGTQVTVSS < 22C51/1- , SEQ ID NO: 2164 ; PRT; ->EVQLVESGGGLVQAGASLRLSCAASGRTFSRYDI 121GWFRQAPGKGREFVAAINWSGGTTSFGDSVKGRF TISRDNAKNTVYLQMNSLKPEDTAVYYCAALRSWPRGVDSGSWGQGTQVTVSS < 12D11/1- , SEQ ID NO: 2165 ; PRT; ->EVQLVESGGGLVQTGGSLRLSCAASGRTFSGSRM 123GWFRQAPGKEREFVAAIRWSGGITWYAESVKSRF TISRDNTKNTIDLQINSLKPEDTAVYYCAADVIYKNIGSGSFDYWGQGTQVTVSS < 12D14/1- , SEQ ID NO: 2166 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSGSRM 123GWLRQAPGKEREFVAAVRWSGGITWYAESVKGRF TISRDNTKNTIDLQINSLKPEDTAVYYCAADVIYKNIGSGSFDYWGQGTQVTVSS < 12C111/1- , SEQ ID NO: 2167 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAVSGLTFSSYAM 123GWFRQAPGKVREFVATISRSGGRTSYADSVKGRF IVSRDNAKNTADLQMNDLKPEDTAVYYCGASKWYGGFGDTDIEYWGQGTQVTVSS < 22B55/1- , SEQ ID NO: 2168 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAVSGLTFSTYAM 123GWFRQAPGKVREFVATISRSGGRTSYADSVKGRF IVSRDNAKNTADLQMNELKPEDTAVYYCGASKWYGGFGDTDIEYWGQGTQVTSS < 14H121/1- , SEQ ID NO: 2169 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGITFRFKAM 113GWFRQGPGKRRELVARIAGGSTNYADSVKGRFTI SRDDAKNTVFLQMNSLKPEDTAVYYCNVDGPFGNWGQGTQVTVSS < 12C71/1- , SEQ ID NO: 2170 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCTASGGTFGSYAL 125GWFRQSPGKERESVAAIDWDGSRTQYADSVKGRF TISRENVKDTMYLQMNSLQAEDTGVYYCVRSRHSGNTLSFSLKYDYWGQGTQVTVSS < 21A31/1- , SEQ ID NO: 2171 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASEPTFSSVAM 125GWFRQGPGKEREFAATITWSGDSTYVTDSVKGRF TISRDNARNTAYLQMDSLRPEDTAVYSCAARRWSGTLSLFDNEYYYWGQGTQVTVSS < 12C91/1- , SEQ ID NO: 2172 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASGRTSSYYHM 121AWFRQAPGKEREFIAAINLSSGSTYYPDSVKGRF TISRGNAKNTVNLQMNSLKPEDTAVYYCAADNYRDSYLEYDYWGQGTQVTVSS < 14H81/1- , SEQ ID NO: 2173 ; PRT; ->EVQLVESGGGLVQAGGSLSLSCAASGRTFSNYRM 125AWFRQAPRKEREFVAAISRSGESTYFADSMKGRF TISRDNTESTGYLQMNNLKPEDTAVYYCAASWDHGDYVDGGFFYDYWGQGTQVTVSS < 12C42/1- , SEQ ID NO: 2174 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRYAM 124HWFRQAPGSERDFVAGISWDGGSTFYANSVKGRF TISRDNAKNMVYLQMNSLKPEDTAVYYCAAAGSAGPPSIDRQYDYWGQGTQVTVSS < 12D102/1- , SEQ ID NO: 2175 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGSSLSFNAM 118GWSREAPGKRRELVARIISDDSTLYADSVKGRFT ISRDYAKNTAYLQMNSLKPEDTAVYYCVADVRDSIWRSYWGQGTQVTVSS < 11A52/1- , SEQ ID NO: 2176 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRALSNYAM 120RWFRQAPGKEREFVATINWSGSHTDYRDSVKGRF TISRDNAENTVYLQMNSLTPEDTAVYYCASGWGATQAQSGFWGQGTQVTVSS < 14H111/1- , SEQ ID NO: 2177 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRALISFAM 120RWFRQAPGKEREFVAAINWSGTHTDYADSVKGRF TISRDNAENTVYLLMNSLIPEDTAVYYCATGWGATQAQHGFWGQGTQVTVSS < 11B61/1- , SEQ ID NO: 2178 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTSSGYGM 120GWFRQAPGKEREFVAAVGWYGSTYFADSVKGRFT IYRDNAQNTMYLQMNSLKPEDTAVYYCAASSSLATISQPSSWGQGTQVTVSS < 12E42/1- , SEQ ID NO: 2179 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAHSGRAFSLRTM 118GWYRQAPGNQRELVALISAGDSTYYPDSVKGRFT VSRDNAKNTVYLQMNSLKPEDTAVYYCNAKAVTSRDHEYWGQGTQVTVSS < 13F81A/1- , SEQ ID NO: 2180 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFSRYAM 128GWFRQAPGKEREFVAAISWTGGSSYYGDSVKGRS TISRENAENTVYLQMNSLKPEDTAVYYCAANSDEFYSGTLKLQSRMVEYWGQGTQVTVSS < 11B102/1- , SEQ ID NO: 2181 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGGIFSSHAI 118SWFRQAFGKAREFVAAINWSGSHRDYADSAKGRF TISRDNAKKTAYLQMNSLRPEDTAVYYCVGGWKTDEYVKWGQGTQVTVSS < 21A41/1- , SEQ ID NO: 2182 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRIFSNYAW 120SWFRQAPGKERGFVAAINWSGGYTDYADSVKGRF TISRDNTKNTVYLQMNSLKPEDTAVYYCRPGWVTPSYEYGNWGQGTQVTVSS < 14H101/1- , SEQ ID NO: 2183 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFISSPM 128GWFRQAPGKEREVVAATTRSGGLPYYSDSVKGRF TISRDNAKNTVDLQMSSLKPEDTAAYYCAADQKYGMSYSRLWLVSEYEYWGQGTQVTVSS < 12E21/1- , SEQ ID NO: 2184 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGSIDSIHVV 115GWYRKAPGKQREVVAYIGTAGATHYADSVKGRFT ISRDNAENLVYLQMNNLKPEDTAVYYCSAGWGDSAYWGQGTQVTVSS < 13F21/1- , SEQ ID NO: 2185 ; PRT; ->EVQLVESGGGLVQSGGSLRLSCVASGTIVSINAT 123SWYRQAPGNQRELVATIIGDGRTHYADSVKDRFT ISRDAAANLVYLQMNSLKPSDTAIYSCNANGIESYGWGNRHFNYWTVGTQVTVSS < 12E33/1- , SEQ ID NO: 2186 ; PRT; ->EVQLVESGGGMVQAGGSLRLSCAASGLTLSNYGM 119GWFRQAPGKEREFVSSINWSGTHTYDADFVKGRF IISRDNAKNTVYLQINSLKPEDTAVYYCAAGGWGTGRYNYWGQGTQVTVSS < 13G11/1- , SEQ ID NO: 2187 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFISNYA 122MGWFRQAPGKEREFVATINWSGSHSDYADSVKGR FTISRDNAKNTVYLQMNNLKSEDTAVYYCAPGWGTAPLSTSVYWGQGTQVTVSS

TABLE B-3 Nanobodies against HER2 obtained as described in Example 4 ;PRT < Name , SEQ ID NO: # (protein) Amino acid sequence< 118N121_A1_4_OK/ , SEQ ID NO: 1988 ; PRT; ->EVQLVESGGGFVQTGGSPRLSCAASGRSFSEYAA 1- AWFRQSPGKERDLVAGIMWDGRSLFYADSVKGRF127 TISRDNAKNTLHLQMNSLKPEDTAVYYCAYHKTP YTTLELNRPHAFGSWGQGTQVTVSS< 118N121_A6_2_OK/ , SEQ ID NO: 2188 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASGRTFSGYSV 1- GWFRQSPGKEREFVGGINWSGRTYYVDSVKGRFT123 FSRDNAKNTVYLQMNSLKPEDTAIYLCAVDRFNT IANLPGEYDYWGQGTQVTVSS< 118N121_B8_1_OK/ , SEQ ID NO: 2189 ; PRT; ->EVQLVESGGGLVQDGGSLRLSCAASGQLANFASY 1- AMGWFRQAPGKAREFVAAIRGSGGSTYIADPARS135 TYYADFVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCACETFNSISNLPGEYDYWGQGTQVTVSS< 118N121_A2_2_OK/ , SEQ ID NO: 2190 ; PRT; ->KVQLVESGGGLVQAGGSLRLSCAASGRTFSNYSV 1- GWFRQAPGKEREFVAALSKDGARTYYAASVKGRF124 TIYRDNAKNVVYLQMSVLNGEDTAVYYCAADHFT FMSNLPSEYDYWGQGTQVTVSS< 118N121_A8_2_OK/ , SEQ ID NO: 2191 ; PRT; ->EVQLVESGGGLVQAGGSLTLSCVISGLTLESHAM 1- GWFRQAPGEEREFVATIRWSGSATFYSDSVKGRF124 TISRDNAKNTVYLQMNSLKPEDTAVYYCAARKIY RSLSYYGDYDSWGQGTQVTVSS< 118N121_B3_1_OK/ , SEQ ID NO: 2192 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGRTFSDLAL 1- GWFRRAPGKEREHVAAISSSGVTTIYADSVRGRF123 TISRDEAKNTVYLEMNSLKTDDTAVYYCAARLTM ATPNQSQYYYWGQGTQVTVSS< 118N121_A5_2_OK/ , SEQ ID NO: 2193 ; PRT; ->EVQLVESGGGSVQPGGSLRLSCVASGSISSVNAM 1- GWHRQVSGKERELVAIVTDGFTNYADFAKGRFTI114 SRDNAKTTVYLQMNSLQPEDTARYYCRYSGIGTD NWGQGIEVTVSS < 118N121_A9_2_OK/ ,SEQ ID NO: 2194 ; PRT; -> EVQLVESGGGSVQPGGSLRLSCVASGSISSVNAM 1-GWHRQVPGKQRELVAIVTDGFTNYADFAKGRFTI 114SRDNAKTTVYLQMNSLQPEDTARYYCRYSGIGTD NWGQGIEVTVSS < 118N121_A7_1_OK/ , SEQID NO: 2195 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGNIKSIDVM 1-GWHRQAPGKERELVSDISFGGNTNYANSVKGRFT 122ISRDNAKNTVYLQMNSLKPEDTAVYYCYADILYK TDIYYRNDFWGQGTQVTVSS< 118N121_A10_1_OK/ , SEQ ID NO: 2196 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGFSFADYAI 1- GWFRQAPGKEREGVSCIANSEGTKYYADSAQGRL131 PISSDNAKKTVYLQMDSLKPEDTAVYYCAALPYT ICPVVVKKGAVYYGVDDYWGKGTQVTVSS< 118N121_A11_1_OK/ , SEQ ID NO: 2197 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFPFGMYGM 1- RWVRQAPGKGPERVSSINSDGDTTYYADSVKGRF120 TISRDNDENMLYLQMNSLKPEDTAVYYCATGFSD RSFAVTHKGQGTQVTVSS< 118N121_B7_4_OK/ , SEQ ID NO: 2198 ; PRT; ->EVQLVESGGGLEQAGGSLRLSCAASGLTFRSAAM 1- GWFRQGPGKEREFVAAISRDGAATYYTDSVKGRF124 TISRDNAKNTVFLQMNSLKPEDTAIYYCAADFRL ARLRVADDYDYWGQGTQVTVSS< 118N121_B2_1_OK/ , SEQ ID NO: 2199 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGFSLDDRAI 1- AWFRQAPGKAREGVSCITPHHGGIIFTRESVKGR130 FATSSDSAKNTVYLQMHSLKPEDTAVYYCATLRT DYSINWANCQRDSLYGYWGQGTQVTVSS< 118N121_B7_1_OK/ , SEQ ID NO: 2200 ; PRT; ->EMQLVESGGGLVQPGGSLRLSCAASGNIPPINAM 1- AWYRQAPGNERELVAAVTSGGGTNYATSVKGRFI119 ISRDDSKNTVDLQMNSLKPEDTAVYYCNLGGWTR THPFDYWGQGTQVTVSS

TABLE B-4 Bivalent Nanobodies against HER2 as described in Example 12 ;PRT < Name , SEQ ID NO: # (protein) Amino acid sequence < 2A4-9GS-2A4 ,SEQ ID NO: 2201 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSAINWSGSHRNYADSV KGRFTISRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQSTTKNQGYWGQGTQVTVSSGGGGSGGG SEVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSAINWSGSHRNYADS VKGRFTISRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQSTTKNQGYWGQGTQVTVSS < 2A5-9GS-2A5 , SEQ ID NO: 2202 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCATSGFTFDDY AMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYC AKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCATSGF TFDDYAMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDT AVYYCAKNWGDAGTTWFEKSGSAGQGTQVTVSS< 2C3-9GS-2C3 , SEQ ID NO: 2203 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDY GMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYC NQGWKIVPTDRTGHGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCVASGFSLDDYG MTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYCN QGWKIVPTDRTGHGTQVTVSS < 2D3-9GS-2D3 ,SEQ ID NO: 2204 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSV KGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGG SGGGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTD YADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 5F7-9GS-5F7 , SEQ ID NO: 2205 ; PRT;-> EVQLVESGGGLVQAGGSLRLSCAASGITFSIN TMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGITFSINT MGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKR FRTAAQGTDYWGQGTQVTVSS

TABLE B-5 Bispecific Nanobodies against HER2 and against serum albuminas described in Example 12 ; PRT < Name , SEQ ID NO: # (protein) Aminoacid sequence < 2C3-9GS- , SEQ ID NO: 2206 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDY ALB1 GMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLRSEDTAVYYC NQGWKIVPTDRTGHGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFG MSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCT IGGSLSRSSQGTQVTVSS < 2A4-9GS- , SEQ IDNO: 2207 ; PRT; -> EVQLVESGGGLVQPGGSLRLSCAASGFIFDDY ALB1AMSWVRQAPGKGLEWVSAINWSGSHRNYADSV KGRFTISRDNAKKTVYLQMNSLQSEDTAVYYCGTGWQSTTKNQGYWGQGTQVTVSSGGGGSGGG SEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADS VKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS < 2A5-9GS- , SEQ ID NO: 2208 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCATSGFTFDDY ALB1 AMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYC AKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGF TFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDT AVYYCTIGGSLSRSSQGTQVTVSS < 2D3-9GS- ,SEQ ID NO: 2209 ; PRT; -> EVQLVESGGSLVQPGGSLRLSCAASGFTFDDY ALB1AMSWVRQVPGKGLEWVSSINWSGTHTDYADSV KGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGG SGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS < 5F7-9GS- , SEQ ID NO: 2210 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGITFSIN ALB1 TMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFG MSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCT IGGSLSRSSQGTQVTVSS

TABLE B-6 Biparatopic Nanobodies against HER2 ; PRT < Name , SEQ ID NO:# (protein) Amino acid sequence < 27B3-35GS- , SEQ ID NO: 2211 ; PRT; ->EVQLVESGGGLVQAGGSLTLSCTASETTVRIR 2D3 TMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCR DINRDIWGQGSQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNAN NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27C3-35GS- , SEQ ID NO: 2212 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFDDY 2D3 ATSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYC ARPRGSSLYLLEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQ LVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGR FTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27E5-35GS- , SEQ ID NO: 2213 ; PRT; ->EVQLVESGGGLVQAGGSLTLSCTASETTVRIR 2D3 TMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCR DINRDIWGQGSQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNAN NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27F2-35GS- , SEQ ID NO: 2214 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGIISSFR 2D3 TLAWYRQAPGKQRDWVATISSAGGTAYADAVKGRFTISISRDNVEYTVDLQMDSLKPEDTAVYY CRDINGDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGS LVQGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27D6-35GS- , SEQ ID NO: 2215 ; PRT; ->EVQLVESGGGLVQAGGSLTLSCTASETTVRIR 2D3 TMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCR DINRDIWGQGSQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNAN NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 30D10-35GS- , SEQ ID NO: 2216 ; PRT; ->EVQLVESGGGLVQAGGSLTLSCTASETTVRIR 2D3 TMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCR DINRDIWGQGSQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNAN NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 47D5-35GS- , SEQ ID NO: 2217 ; PRT; ->KVQLVESGGGLVQGGSLRLSCAASGSIFGFN 2D3 DMAWYRQAPGKQRELVALISRVGVTSSADSVKGRFTISRVNAKDTVYLQMNSLKPEDTAVYYCY MDQRLDGSTLAYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE SGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTI SRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQTQVTVSS < DUMMY-35GS- , SEQ ID NO: 2218 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSY 2D3 PMGWFRQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSC AAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADS VKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 2D3-35GS- , SEQ ID NO: 2219 ; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDY 2D3 AMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYC AKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVK GRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < DUMMY-35GS- , SEQ ID NO: 2220 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGRTFRSY 47D5 PMGWERQAPGKEREFVASITGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYSC AAYIRPDTYLSRDYRKYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG SEVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSADSV KGRFTISRVNAKDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSS < 5F7-35GS- , SEQ ID NO: 2221 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGITFSIN 47D5 TMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCK RFRTAAQGTDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES GGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSADSVKGRFTISR VNAKDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSS < 47D5-35GS- , SEQ ID NO: 2222 ; PRT; ->KVQLVESGGGLVQPGGSLRLSCAASGSIFGFN 5F7 DMAWYRQAPGKQRELVALISRVGVTSSADSVKGRFTISRVNAKDTVYLQMNSLKPEDTAVYYCY MDQRLDGSTLAYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE SGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTIS RDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSS < 2D3-35GS- , SEQ ID NO: 2223 ; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDY 47D5 AMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYC AKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSADSVKG RFTISRVNAKDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSS < 27F7-35GS- , SEQ ID NO: 2224 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVVSGIPSTIR 2D3 AMAWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRFTISSDNAKKTIYLQMNSLKPDDTAVYYC RDVNREYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSL VQPGGSLRLSCAASGFTEDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNA NNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 28F6-35GS- , SEQ ID NO: 2225 ; PRT; ->EVQLVESGGGLVQAGGSLNLSCVASGIPFSTR 2D3 TMAWYRQPPGNERDWVATIRSGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLEPEDTATYYCWD VNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQ PGGSLRLSCAASGFTFDDYAMSWVRQVRGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANN TLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 28G3-35GS- , SEQ ID NO: 2226 ; PRT; ->EVQLVESGGGLVQAGGSLNLSCVASGIPFSTR 2D3 TMAWYRQPPGNERDWVATIRSGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLEPEDTATYYCWD VNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQ PGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANN TLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 28G5-35GS- , SEQ ID NO: 2227 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVTSRRPASTR 2D3 TMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCR DVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNAN NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEYSGSAGQGTQVTVSS < 29D9-35GS- , SEQ ID NO: 2228 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASRIPFSTR 2D3 TMAWYRQAPGKQRDWVATIGTSGPPRYADSVKGRFTVSRDNAKNTVYLQMNSLKAEDTAVYYCW DVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNAN NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 29E9-35GS- , SEQ ID NO: 2229 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASRIPASIR 2D3 TMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCR DVNGDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV QPGGSLRLSCAASGFTEDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNAN NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 30E10-35GS- , SEQ ID NO: 2230 ; PRT; ->KVQLVESGGSLVPPGGSLRLSCAASGFTFDDY 2D3 AMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYC AKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVK GRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 31D11-35GS- , SEQ ID NO: 2231 ; PRT; ->EVQLVESGGGLVQAGGSLNLSCVASGIPFSTR 2D3 TMAWYRQPPGNERDWVATIRSGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLEPEDTATYYCWD VNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQ PGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANN TLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27G2-35GS- , SEQ ID NO: 2232 ; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDY 2D3 AMTWVRQTPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSDDTAVYYC AKNWGDAGTTWFEKSGSAGPGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGSLVQPGSSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVK GRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < P27G4-35GS- , SEQ ID NO: 2233 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVTSRRPASTR 2D3 TMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCR DVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNAN NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27G5-35GS- , SEQ ID NO: 2234 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASRIPASIR 2D3 TMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTVSRDNAKFTVYLQMNSLKPEDTAVYYCR DVNGDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNAN NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27G7-35GS- , SEQ ID NO: 2235 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVTSRRPASTR 2D3 TMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCR DVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNAN NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27H1-35GS- , SEQ ID NO: 2236 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVTSRRPASTR 2D3 TMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCR DVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNAN NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27H2-35GS- , SEQ ID NO: 2237 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVTSRRPASTR 2D3 TMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCR DVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNAN NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27H3-35GS- , SEQ ID NO: 2238 ; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDY 2D3 AMTWVRQASGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYC AKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVK GRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27H4-35GS- , SEQ ID NO: 2239 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASKMTFMRY 2D3 TMGWYRQAPGKQRDLVASIDASGGTNYADSVKGRFTISRDNAKNTVYLEMNSLKPEDTGVYYCN GRWDIVGAIWWGQGTQVTVSSQGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESG GSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISR NNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27H5-35GS- , SEQ ID NO: 2240 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGFTFDDY 2D3 GIGWFRQASGKEREGVSCITSSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYC AALPFVCPSGSYSDYGDEYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSEVQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYA DSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQTVSS < 27H7-35GS- , SEQ ID NO: 2241 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGIAFRIR 2D3 TMAWYRQAPGKQRDWVATSDSGGTTLYADSVKGRFTVSRDNAENTVYLQMNSLKPEDTAVYYGR DVNRDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNAN NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27A3-35GS- , SEQ ID NO: 2242 ; PRT; ->EVQLVESGGGLVQAGGSLSLSCVASGRFFSTR 2D3 VMAWYRQTPGKQREFVASMRGSGSTNYADSVRGRFAISRDNAKNTVYLQMNTLKPEDTAVYYCR DINEDQWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNAN NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27A4-35GS- , SEQ ID NO: 2243 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVTSRRPASTR 2D3 TMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCR DVNADYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNAN NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27A5-35GS- , SEQ ID NO: 2244 ; PRT; ->EVQLVESGGGLVQAGGSLNLSCVASGIPFSTR 2D3 TMAWYRQPPGNERDWVATIRSGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLEPEDTATYYCWD VNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQ PGGSLRLSCAASGFTEDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANN TLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27B1-35GS- , SEQ ID NO: 2245 ; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDY 2D3 AMSWVRQAPGKGLEWISSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNNLKFEDTAVYYC AKNWRDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVK GRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27B2-35GS- , SEQ ID NO: 2246 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCVASGIPSIRA 2D3 IAWYRQAPGKQRDWVATSGTGYGATYDDSVKGRFTLSRDNAKNTVYLQMNSLKPEDTAVYYCRD VNRDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQ PGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANN TLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27B5-35GS- , SEQ ID NO: 2247 ; PRT; ->EVQLVESGGGLVQAGGSLRLPCAASGIAFRIR 2D3 TMAWYRQAPGKQRDWVATSDSGGTTLYADSVKGRFTVSRDNAENTVYLQMNSLKPEDTAVYYCR DVNRDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNAN NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27B7-35GS- , SEQ ID NO: 2248 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY 2D3 AMSWVRQAPGKGLEWVSAISSGGGSITTYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTALYY CAKARSSSSYYDFGSWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQ LVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGR FTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27C2-35GS- , SEQ ID NO: 2249 ; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDY 2D3 AMTWVRQASGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYC AKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVK GRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27C5-35GS- , SEQ ID NO: 2250 ; PRT; ->EVQLVESGGGLVQAGGSLNLSCVASGIPFSTR 2D3 TMAWYRQPPGNERDWVATIRSGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLEPEDTATYYCWD VNGDIWGQGTPVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLVQ PGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANN TLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27C7-35GS- , SEQ ID NO: 2251 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGIAFRIR 2D3 TMAWYRQAPGKQRDWVATSDSGGTTLYADSVKGRFTVSRDNADNTVYLQMNSLKPEDTAVYYCR DVNRDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGSLV QPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNAN NTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27D1-35GS- , SEQ ID NO: 2252 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDY 2D3 GMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYC NQGWKILPAERRGHGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES GGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTIS RNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27D2-35GS- , SEQ ID NO: 2253 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCAASGLGIAFS 2D3 RRTMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGRFTISRDNAKNTVYLQVNSLKPEDTAVYY CWDTNGDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGS LVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27D3-35GS- , SEQ ID NO: 2254 ; PRT; ->EVQLMESGGGLVQPGGSLRLSCAASGLGIAFS 2D3 RRTMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGRFTISRDNAENTVYLQMNSLKPEDTAVYY CWDVNRDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGS LVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISRNN ANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27D4-35GS- , SEQ ID NO: 2255 ; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDY 2D3 AMTWVRQASGKGLEWVSSINWSGTHTDYADSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYC AKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVK GRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27D7-35GS- , SEQ ID NO: 2256 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDY 2D3 GMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYC NQGWKILPTNRGSHGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES GGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTIS RNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27E2-35GS- , SEQ ID NO: 2257 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDY 2D3 GMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLTPEDTAVYYC NQGWKIIPTDRRGHGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES GGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTIS RNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27E4-35GS- , SEQ ID NO: 2258 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGSTFSIN 2D3 RMAWYRQSPGKQRELVAAVDNDDNTEYSDSVAGRFTISRDNAKNAVHLQMNSLRLEDTAVYYCN AKQLPYLQNFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESG GSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISR NNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27E7-35GS- , SEQ ID NO: 2259 ; PRT; ->EVQLVESGGGLVQAGGSLRLSCAASGITFRRY 2D3 DMGWYRQFPGKERELVATILSEGDTNYVDPVKGRFTISRDNAKNTVYLQMNDLKPEDTAVYYCN GVWRAIGRTYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESG GSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTISR NNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 29H1-35GS- , SEQ ID NO: 2260 ; PRT; ->EVQLVESGGSLVPPGGSLRLSCAASGFTFDDY 2D3 AMSWVRQAPGKGLEWVSSINWSGTHTGYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYC AKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVK GRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 30H9-35GS- , SEQ ID NO: 2261 ; PRT; ->EVQLVESGGSLVQPGGSLRLSCAASGFTFDDY 2D3 AMTWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRNNANNTLYLQMNSLKSEDTAVYYC AKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVK GRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 39C1-35GS- , SEQ ID NO: 2262 ; PRT; ->EVQLVESGGSLVPPGGSLRLSCAASGFTFDDY 2D3 GMSWVRQAPGKGLEWVSSINWSGTHTDYTDSVKGRFTISRMNANNTLYLQMNSLKSEDTAVYYC AKNWGDAGTTWFEKSGSAGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE VQLVESGGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVK GRFTISRNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 27G8-35GS- , SEQ ID NO: 2263 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDY 2D3 GMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAYNTLFLQMNSLTPEDTAVYYC NQGWKILPAERRGHGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES GGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTIS RNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 29H2-35GS- , SEQ ID NO: 2264 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVASGFSLDDY 2D3 GMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNNLTPEDTAVYYC NQGWKIIPTDRRGHGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES GGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTIS RNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS < 38C6-35GS- , SEQ ID NO: 2265 ; PRT; ->EVQLVESGGGLVQPGGSLRLSCVGSGFSLDDY 2D3 AMTWVRQAPGKGLEWVSSINWSGTHTDYADSVKGRFTISRDNAKNTLFLQMNSLSPEDTAVYYQ NQGWKIRPTIPMGHGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVES GGSLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSSINWSGTHTDYADSVKGRFTIS RNNANNTLYLQMNSLKSEDTAVYYCAKNWRDAGTTWFEKSGSAGQGTQVTVSS

TABLE C-1 Sequence of HER2 binding Nanobodies aligned by familyHERCEPTIN ® COMPETING 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_A1_4_OK/EVQLVESGGGFVQTGGSPRLSCAASGRSFSEYAAAWFRQSPGKERDLVAGIMWDGRSLFYADSVKGRFTISRDNAKNTLHLQMNSLKPEDTAVYYCAYHKTPYTTLELNRPHAFGSWGQGTQVTVSS1-127 OT-FAB COMPETING 47D5KVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSADSVKGRFTISRVNAKDTVYLQMNSLKPEDTAVYYCYMDQRLDGSTLAYWGQGTQVTVSSHER2 BINDING 14B11EVQLVESGGGLVQAGGSLRLSCAASGSTFSSYGMGWFRQVPGKEREFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTARYYCGVETYGSGSSLMTEYDYWGQGTQVTVSS14B10EVQLVESGGGLVQAGGSLRLSCAVNSRTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSIKGRFTISRDNAKETVYLQMNSLKPDDTGVYYCAAETYGSGSSLMSEYDYWGQGTVTVSS14B4EVQLVESGGGLVQAGGSLRLSCAVSSRAFSSYGMGWFRQAPGKDREFVATINWSGVTAYADSIKGRFTISRDNAKETVYLQMNSLKPEDTGVYYCAAETYGSGSSLMSEYDYWGQGTQVTVSS14C11EVQLVESGGGLVQAGGSLRLSCAVNSRTFSSYGMGWFRQAPGKEREFVATINWSGATAYADSIKGRFTISRDNAKETVYLQMNSLKPDDTGVYYCAAETYGSGSSLMSEYDYWGQGTQVTVSS14B5EVQLVESGGGLVQAGGSLRLSCAVSSRAFSSYGMGWFRQAPGKDREFVATINWSGVTAYADSIKGRFTISRDNAKETVYLQMNSLKPDDTGVYYCAAETFGSGSSLMSEYDYWGQGTQVTVSS14C6EVQLVESGGGSVQAGGSLRLSCVASEGTFSSYGMGWFRQAPGKERAFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCATDTYGSGSSLMNEYDYWGQGTQVTVSS14A4EVQLVESGGGSVQAGSSLTLSCVASEGTFSSYGMGWFRQAPGKERAFVATINWSGVNAYADSVKGRFTISRDNAKKTAYLQMNSLKPEDTAVYYCAAETYGSGSSLMNEYDYWGQGTQVTVSS14B3EVQLVESGGGLVQPGGSLTLSCVASEGTFSSYGMGWFRQAPGKERAFVATINWSGVNAYADSVKGRFTISRDNAKKTAYLQMNSLKPEDTAVYYCAAETYGSGSSLMNEYDYWGQGTQVTVSS14C1EVQLVESGGGSVQAGGSLRLSCAASGSTFSSYGMGWFRQAPGKERAFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCATETYGSGSSLMNEYDYWGQGTQVTVSS14A12EVQLVKSGGGLVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLISEYDYWGHGTQVTVSS14A2EVQLVESGGGLVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLISEYDYWGHGTQVTVSS14A1EVQLVESGGGSVQAGGSLRLSCAASERTFSSYGMGWFRQAPGKEREFVATINWSGVTAYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAAEPYGSGSSLMSEYDYWGHGTQVTVSS17C3EVQLVESGGGLVQAGGSLRLSCAANGLTFRRYDMGWYRQAPGQQREWVAAISGAGDINYADSVKGRFTMARDNANHTVHLQMNSLKPEDTAVYYCNANWKMLLGVENDYWGQGTQVTVSS46D3KVQLVESGGGLVQAGGSLRLSCAASGRTFTEYSMGWFRQAPGKEREFVATISWNYGYTYYSDSVKGRFTVGRDIAENTVYLQMNTLKSEDTAVYYCAAKIGWLSIRGDEYEYWGQGTQVTVSS27H5EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYGIGWFRQASGKEREGVSCITSSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAALPFVCPSGSYSDYGDEYDYWGQGTQVTVSS 17C2EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYAMSWVRQAPGKGLEWVSAVDSGGGRTDYAHSVKGRFTISRDNAKNTLYLQMSSLKPEDTALYYCTKHVSDSDYTEYDYWGQTQVTVSS17D11EVQLVESGGGLVQAGGSLRLSCTASGRTSSTSAMGWFRQAPGKEREFVATISRGGSATYYADSLKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAARRSSLYTSSNVFEYDYWGQGTQVTVSS 15A6EVQLVESGGGLVQAGGSLRLSCVTTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSS17B6EVQLVESGGGLVQPGGSLRLSCAASRIPFSTRTMAWYRQAPGKQRDWVATIGTSGPPRYADSVKGRFTVSRDNAKNTVYLQMNSLKAEDTAVYYCWDVNADYWGQGTQVTVSS17C5EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAPGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTPVTVSS15E11EVQLVESGGGLVQAGGSLRLSCVASRIPFSSRTMAWYRQAPGKQRDWVATISARGMPAYEDSVKGRFTVSRDNDKNTLYLQMNSLKPEDTAVYYCRDVNADYWGQGTQVTVSS15C2EVQLVESGGGLVQAGGSLRLSCVTSRRPASTRTMAWYRQAQGKQRDWVATISSHGLPVYADSVKGRFTVSRDNANNTVYLQMNTLKPEDTAVYYCRDVNADYWGQGTQVTVSS2A3 EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQAPGKPRDWVATIRNGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLKPEDTATYLCRDVNGDIWGQGTQVTVSS 27A5EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQPPGNERDWVATIRSGAPVYADSVKGRFTVSRDNAKNTLYLQMNSLEPEDTATYYCWDVNGDIWGQGTPVTVSS 2C5EVQLVESGGGLVQAGGSLNLSCVASGIPFSTRTMAWYRQTPGKSRDWVATIRSGTPVYADSVKGRFTVSRDNAKNTLYLRMNSLKSEDSATYTCRAVNADIWGQGTQVTVSS 27G5EvQLVESGGGLVQPGGSLRLSCVASRIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS13A9EVQLVESGGGLVQAGGSLRLSCVASRIPASIRTMAWYRQAPGKQRDWVATIGTGGTPAYADSFKGRFTVSRDNANHTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS29E9EVQLVESGGGLVQPGGSLRLSCVASRIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS15D8EVQLVESGGGLVQPGGSLKLSCVASTIPASIRTMAWYRQTPGNQRDWLATIGSSGTPAYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNGDYWGQGTQVTVSS15G4EVQLVESGGGLVQAGGSLRLSCVASGIPFRSRTMAWYRQAPGKTRDWVATIGTHGTPLYADSVKGRFTVSRDNAKNTLYLQMNSLKPEDTAVYYCWDVNGDYWGQGTQVTVSS15D12EVQLVESGGGLVQAGESLRLSCATSGITFKRYVMGWYRQGPGKQRELVATVNDGGTTSYADSVKGRFAISRDNAKNTAYLQMNSLKAEDTAVYYCNAVWKLPRFVDNDYWGQGTQVTVSS15E12EVQLMESGGGLVQAGGSLRLSCAANGLTFRRYDMGWYRQAPGQQREWVAAISGAGDINYADSVKGRFTMARDNANHTVHLQMNSLKPEDTAVYYCNANWKMLLGVENDYWGQGTQVTVSS13D7EVQLAVESGGGLVQAGGSLRLSCAANGLTFRRYDMGWYRQAPGQQREWVAAISGAGDINYADSVKGRFTMARDNANHTVHLQMNSLKPEDTAVYYCNANWKMLLGVENDYWGQGTQVTVSS13A8EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTV YADSMKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCWDVNRDYWGQGTQVTVSS15A4EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTV YADSMKGRFTISRDNAKNTVYLQINSLKPEDTAVYYCWDVNRDYWGQGTQVTVSS17F7EVQLVESGGGLVQAGGSLRLSCVASGIAQS  IRVMAWYRQPPGKQRDWVGTISSDGTAN YADSVKGRFTISRDNAKKTMYLQMNSLKPDDTAVYYCRDVNRDYWGQGTQVTVSS15C8EVQLVESGGGLVQAGGSLRLSCAASGIAFR  IRTMAWYRQAPGKQRDWVATSDSGGTTL YADSVKGRFTVSRDNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS17A10EVQLVESGGGLVQAGGSLRLSCVASGIPSI   RAIAWYRQAPGKQRDWVATSGTGYGAT YDDSVKGRFTLSRDNAKNTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS27D3EVQLMESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTV YADSMKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCWDVNRDYWGQGTQVTVSS13B12EVQLVESGGGLVQAGGSLRLSCAASGIAFR  IRTMAWYRQAPGKQRDWVATIGSDGTTI YADSVKGRFTLSRHNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS15B2EVQLVESGGGLVQAGGSLRLSCVVSGIPSS  IRAMAWYRQAPGRQRDWVATIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLKPDDTAVYYCRDVNRDYWGQGTQVTVSS15B11EVQLVESGGGSVQAGGSLRLSCVVSGIPSS  IRAMAWYRQAPGRQRDWVATIYSRSGGAVYADSVKGRFTISSDNAKNTIYLQMNSLKPDDTAVYYCRDVNRDYWGQGTQVTVSS13C9EVQLVESGGGLVQAGGSLRLSCVASGIPSI  HAMAWYRQAPGKQRDWGATTYSRGG  TTYNDSAKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS17D5EVQLVESGGGLVQPGGSLRLSCAASGIIGT  IRTMAWYRQAPGKQRDWVA  SIGTRGAPVYADSVNGRFTISRDGATNTVFLQMNNLKPEDTAVYYCRDVNRDYWGQGTQVTVSS27B5EVQLVESGGGLVQAGGSLRLPCAASGIAFR IRTMAWYRQAPGKQRDWVA  TSDSGGTTLYADSVKGRFTVSRDNAENTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS27C7EVQLVESGGGLVQAGGSLRLSCAASGIAFR IRTMAWYRQAPGKQRDWVA  TSDSGGTTLYADSVKGRFTVSRDNADNTVYLQMNSLKPEDTAVYYCRDVNRDYWGQGTQVTVSS13D4EVQLVESGGGLVQAGCSLRLSCVVSGIPSS IRAMAWYRQAPGRQRDWVA TIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLEPDDTAVYYCRDVNREYWGQGTQVTVSS15G5EVQLVESGGGLVQAGGSLRLSCVVSGIPST IRAMAWYRQAPGRQRDWVA TIYSPSGSAVYADSVKGRFTISSDNAKKTIYLQMNSLKPDDTAVYYCRDVNREYWGQGTQVTVSS13C4EVQLVESGGGLVQAGGSLRLSCVVSGIPSS IRAMAWYRQAPGRQRDWVA TIYSPSGSAVYADSVKGRFTISSDNAKSTIYLQMNSLKPDDTAVYYCRDVNREYWGQGTQVTVSS46G1EVQLVESGGGLVQAGGSLRLSCAASGRTFSDDAMGWFRQAPGKERECVASLYLNGDYPYYADSVKGRFTISRDNAKNAVILQMNNLKTEDTAVYYCAAKPGWVARDPSQYNYWGQGTQVTVSS46E4EVQLVESGGGLVQAGGSLRLSCAASGRAFKDDAVGWERQAPGKERECVASMYLDGDYPYYADSVKGRFTISRDNAKNAVILQMNNLKTEDTAVYYCAAKPGWVARDPSEYNYWGQGTQVTVSS17B5EVQLVESGGGLVQTGGSLRLSCAASGSTFRTDMMGWYRQAPGKQREFVASITKFGSTNYADSVKGRFTISNDNAKDTVYLQMNSLKSEDTAVYYCRNFNRDLWGQGTQVTVSS15C9EVQLVESGGGLVQAGGSLKLSCVNSGIPSTLRAMAWYRQAPGRQRDWVATSSNTGGTTYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCRDVNRDLWGQGTQVTVSS13D10EVQLVESGGGLQPGGSLRLSCAASSVITLDSNAIGWERQAPGKEREEVSCIASSDGSTYYAESVKGRFTISKDYTRNTVYLQVNSLKPEDTAVYHCATDANPNCGLNVWNSWGQGTQVTVSS17C6EVQLVESGGGLVQAGGSLTLSCAASGSTSSLDIMAWYRQAPEKQRELVASVSGGGNSDYASSVKGRFTISGDTAKSTLYLQMNSLKPEDTAMYYCYGRDYYYMPFWGQGTQVTVSS15A2EVQLVESGGGLAQAGGSLSLSCAASGRFFS  TRVMAWYRQTRGKQREFVASMRGSGSTNYADSARGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCRDINEDQWGQGTQVTVSS17A8EVQLVESGGGLVQAGGSLSLSCAASGRFFS  TRVMAWYRQTPGKQREFVASMRGSGSTNYADSVRGRFAISRDNAKNMVYLQMNTLKPEDTAVYYCRDINEDQWGQGTQVTVSS15G10EVQLVESGGGLVQAGGSLSLSCAASGRFFS  TRVMAWYRQTPGKQREFVASMRGSGSTNYADSARGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCRDINEDQWGQGTQVTVSS27A3EVQLVESGGGLVQAGGSLSLSCVASGRFFS  TRVMAWYRQTPGKQREFVASMRGSGSTNYADSVRGRFAISRDNAKNTVYLQMNTLKPEDTAVYYCRDINEDQWGQGTQVTVSS17H10EVQLVESGGGLVQAGGSLSLSCSASGRFFS  TRVMAWYRQTPGNQREFVATIHSSGSTIYADSVRGRFAISRDNAKNTVYLQMRSLKPEDTAVYYCRDINADQWGQGTQVTVSS30D10EVQLVESGGGLVQAGGSLTLSCTASETTVR  IRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS15H4EVQLVESGGGLVQAGGSLTLSCAPSESTVS  FNTVAWYRQAPGEQREWVATISRQGMSTYPDSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCRDINHDIWGRGSQVTVSS17B7EVQLVESGGGLVQAGGSLRLSCAASGIISS  FRTMAWYRQAPGKQRDWVATIGSDGLANYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYFCRDINRDYWGQGTQVTVSS15D2EVQLVESGGGLVQAGGSLRLSCVVSGVFGP  IRAMAWYRQAPGKQRDWVATIGSSGHPVYTDSVKGRFTFSKDGAKNTVYLQMNSLKPEDTAVYYCRDINRDYWGQGTQVTVSS17G5EVQLVESGGGLVQPGGSLRLSCAASGIGIAFSSRTMAWYRQAPGKQRDWVATIGSGGTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDYWGQGTQVTVSS15B6EVQLVESGGGLVQPGGSLRLSCAASGIIGS  FRTMAWYRQAPGNQRDWVATIGSAGLASYADSVRGRFTLSRDNAKKTVYLQMNSLKPEDTAIYYCRDINGDYWGQGTQVTVSS27F2EVQLVESGGGLVQAGGSLRLSCAASGIISSFRTLAWYRQAPGKQRDWVATISSAGGTAYADAVKGRFTISISRDNVEYTVDLQMDSLKPEDTAVYYCRDINGDYWGQGTQVTVSS17F5EVQLVESGGGLVQPGGSLRLSCAASGLGIAFSRRTMAWYRQAPGKQRDWVATIAGDGSTVYADSMKGRFTISRDNAKNTVYLQVNSLKPEDTAVYYCWDTNGDYWGQGTQVTVSS17B2EVQLVESGGGLVQPGGSLRLSCAGSGFTFSNYAMTWVRQAPGKGLEWVSGVGGDGVGSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCTKDISTFGWGPFDYWGQGTQVTVSS27H4EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGWYRQAPGKQRDLVASIDASGGTNYADSVKGRFTISRDNAKNTVYLEMNSLKPEDTGVYYCNGRWDIVGAIWWGQGTQVTVSS13A4EVQLVESGGGLVQAGGSLRLSCVASKMTFMRYTMGWYRQAPGKQRDLVASIDSSGGTNYADSVKGRFTISRDNAKNTVYLEMNSLKPEDTGVYYCNGRWDIVGAIWWGQGTQVTVSS2A1EVQLVESGGGLVQAGGSLRLSCVASKITFRRYIMDWYRQAPGKQRELVASINSDGSTGYTDSVKGRFTISRDNTKNTLDLQMNSLKPEDTAVYYCHGRWLEIGAEYWGQGTQVTVSS15E10EVQLVESGGGLVQAGGSLKLSCVASGITFFRYTMGWYRQAPGKERELVAEISSADEPSFADAVKGRFTISRDNAKNTVVLQMNGLKPEDTAVYYCKGSWSYPGLTYWGKGTLVTVSS27E7EVQLVESGGGLVQAGGSLRLSCAASGITFRRYDMGWYRQFPGKERELVATILESEGDTNYVDPVKGRFTISRDNAKNTVYLQMNDLKPEDTAVYYCNGVWRAIGRTYWGQGTQVTVSS47E5EVQLVESGGGLVQAGGSLRLSCAASASIFGFDSMGWYRQAPGNERILVAIISNGGTTSYRDSVKGRFTIARDNAKNTVSLQMNSLKPEDTAVYYCNLDRRSYNGRQYWGQGTQVTVSS2G4EVQLVESGGGLVQAGGSLRLSCAASGNIFSHNAMGWYRQAPGKQRELVTYITINGIANYVDSVKGRFTISRDNTKNTMYLQMVSLKPEDTAVYYCNVGGREYSGVYYYREYWGQGTQVTVSS14D4EVQLVESGGGLVQAGDSLRLSCAASGRALDTYVMGWFRQAPGDGREFVAHIFRSGITSYASSVKGRFTISRDNAKNTVYLQMASLKPEDTAAYYCAARPSDTTWSESSASWGQGTQVTVSS17A5EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYSMSWVRQATGKGLEWVSGISWNGGSTNYADSVKGRFTISRDNVKNTLYLQMNSLKSEDTAVYYCAKDLGNSGRGPYTNWGQGTQVTVSS15D10EVQLVESGGGLVQPGGSLKLSCAASGFTFSSYRMYWVRQAPGKGLEWVSAIKPDGSITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDCGVPGFGWTFSSWGQGTQVTVSS13C2EVQLVESGGGLVQAGGSLRLSCAASGSTFSINRMAWYRQSPGKQRELVAAVDNDDNTEYSDSVAGRFTISRDNAKNAVHLQMNSLRLEDTAVYYCNAKQLPYLQNFWGQGTQVTVSS17G11EVQLVESGGGLVQAGGSLRLSCAASGSTFSINRWGWYRQAPGKQRELVAAIDDGGNTEYSDFVNGRFTISRDNPETAVHLQMNSLKLEDTAVYYCNAKQLPYLQNFWGQGTQVTVSS17A3EVQLVESGGGLVQAGGSLSLSCAASATLHRFDNNWYRQAPGKQRELVATIAHDGSTNYANSVKGRFTISRDNARDTLFLQMHALQPEDTAVYMCNLHRWGLNYWGQGTQVTVSS27B7EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISSGGGSITTYADSVKGRFTISRDNAKNTLYLQMSSLKPEDTALYYCAKARSSSSYYDFGSWGQGTQVTVSS17A6EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISSGGGSITTYADSVKGRFTISTDNAKNTLYLQMSSLKPEDTALYYCAKARSSSSYYDFGSWGQGTQVTVSS17D7EVQLVESGGGLVQPGGSLRLSCAASGFTLDYCAIGWFRQAPGKEREGVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATDRGSGTCYADFGSWGQGTQVTVSS46D4EVQLVESGGGLVQPGGSLRLSCAASGFIFDDYAMSWVRQAPGKGLEWVSSINWSGTHTDYAEDMKGRFTISRDNAKKTLYLQMNSLQSEDTAVYYCAKGWGPAVTSIPVATLGTQVTVSS27B3EVQLVESGGGLVQAGGSLTLSCTASETTV  RIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS27E5EVQLVESGGGLVQAGGSLTLSCTASETTV  RIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS27D6EVQLVEGGGGLVQAGGSLTLSCTASETTV  RIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS30D10EVQLVESGGGLVQAGGSLTLSCTASETTV  RIRTMAWYRQPPGNQREWVATIGSNGFATYPDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRDINRDIWGQGSQVTVSS47G11EVQLVESGGGLVQPGGSLRLSCAASGRIFYPMGWFRQAPGKEREFVAAIGSGDIITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASSRDYSRSRDPTSYDRWGQGTQVTVSS27C3EVQLVESGGGLVQGGSLRLSCAASGFTFDDYATSWVRQAPGKGPEWVSAINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCARPRGSSLYLLEYDYWGQGTQVTVSS

TABLE C-2 k_(off) rate of different Nanobodies as measured in Biacore IDk_(off) (s⁻¹) 2A4 2.05E−03 2A5 1.42E−03 2A6 1.65E−03 2B1 1.55E−03 2C41.26E−03 2D2 1.61E−03 2D4 1.65E−03 2F2 1.65E−03 2F3 1.53E−03 2F51.57E−03 2G5 1.56E−03 2H4 1.61E−03 2B2 1.19E−03 2B3 1.25E−03 2B42.77E−03 2B5 1.15E−03 2C1 1.18E−03 2C2 4.12E−03 2C3 1.11E−03 2D11.27E−03 2D5 1.20E−03 2F1 1.77E−03 2F4 1.07E−03 2G1 1.23E−03 2G21.30E−03 2G3 1.20E−03 2H3 1.09E−03 2H2 1.18E−03 2H3 1.15E−03 2H51.21E−03

TABLE C-3 Overview of k_(d)/k_(off)-, k_(a)-, and K_(d)-values forbinding of Nanobodies 2D3, 5F7 and 47D5 to HER2. Nanobody ID k_(off)(s⁻¹) K_(on) (1/Ms) K_(D) (nM) 2D3 1.48E−03 1.36E+06 1.09 Dummy-2D31.13E−03 1.16E+06 1.77 5F7 3.02E−04 1.02E+06 0.29 47D5 8.62E−04 3.86E+052.23 Dummy-47D5 8.69E−04 2.71E+05 3.21 Fusion of a dummy Nanobody at theN-terminal end of the Nanobodies 2D3 and 47D5 does not significantlyimpact on the binding characteristics of 2D3 or 47D5 respectively.

TABLE C-4 Off-rate analysis of HER2-ECD on 2D3, 47D5 and 2D3-35GS-47D5coated sensor chips Analyte Protein on sensor chip k_(off) (1/s)  100 nMHer2 BCD 2D3-47D5 8.07E−5  100 nM Her2 BCD 2D3 2.10E−3  100 nM Her2 ECD5F7 2.56E−3 1000 nM Her2 ECD 2D3-47D5 5.45E−5 1000 nM Her2 BCD 2D31.51E−3 1000 nM Her2 ECD 5F7 1.31E−3

TABLE C-5 Oligonucleotide primers used for generation of Omnitarg lightchain V_(L) + C_(L) by overlap extension SEQ ID SEQ ID For-sequences NORev-sequences NO >For_LCrescuepAX51 2271 >Rev_LC1pAX51 2283Tgattacgccaagct TAATAACAATCCAGCGGCTGCCGTAGGCAATAGGTATTTCATGTTGAAAATCT >For_LC1pAX51 2272 >Rev_LC2_OT 2284Tgattacgccaagcttgcatgca ATCGCCGACGGACGCGCTCAGGCTACaattctatttcaaggagattttc TCGGAGATTGCGTCATCTGGATGTCG aacatgaGC >For_LC2pAX51_OT 2273 >Rev_LC3_OT 2285 gctggattgttattactcgcggcCCGGCTTCTGTTGATACCAAGCAACC ccagccggccatggccGACATCCCCGATAGATACGTCCTGACTTGCT AGATGACG >For_LC3_OT 2274 >Rev_LC4_OT 2286GCGTCCGTCGGCGATCGCGTTAC CCGCTGAAACGGGAAGGCACACCGGTCATCACATGCAAAGCAACTCAGG GTAACGATATGATGCGGAGTAAAT ACGT >For_LC4_OT2275 >Rev_LC5_OT 2287 ATCAACAGAAGCCGGGCAAGGCT ATAGTAGGTGGCGAAGTCCTCTGGCTCCGAAATTGCTCATTTACTCCGC GCAGGCTAGAGATAGTCAGGGTAA ATCA >For_LC5_OT2276 >Rev_LC6_OT 2288 TTCCCGTTTCAGCGGAAGCGGCT TACCGTACGTTTAATTTCCACTTTCGCGGGTACTGATTTTACCCTGACT TACCCTGGCCAAAGGTATACGGGT ATCT <For_LC6_OT2277 >Rev_LCrescue_VL_OT 2289 TTCGCCACCTACTATTGTCAGCA TCGGAAGGCGGAAAGATACTATATTTACCCGTATACCT TTGG >For_LC7_OT 2278 >Rev_LC7 2290ATTAAACGTACGGTAGCTGCCCC ATACGACGCTGGCCGTACCACTTTTCTAGCGTGTTTATCTTTCCGCCTT AGCTGCTCGTCGGAAGGCGGAAAG CCGA >For_LC82279 >Rev_LC8 2291 CGGCCAGCGTCGTATGTTTACTG CCGGACTGCAGTGCATTATCCACTTTAATAACTTCTATCCGCGCGAAGC CCATTGGACTTTAGCTTCGCGCGG TAAA >For_LC92280 >Rev_LC9 2292 TGCACTGCAGTCCGGCAATTCTC GGTCAGGGTAGAGCTCAGTGAGTAAGAAGAATCCGTGACGGAACAAGAT TGCTATCTTTGCTATCTTGTTCCG AGCA >For_LC102281 >Rev_LC10 2293 AGCTCTACCCTGACCTTGTCAAA GAAAGTCCCTGATGGGTCACTTCACAGGCAGATTATGAAAAACACAAAG GGCGTAAACTTTGTGTTTT TTTA >For_LC112282 >Rev_LC11 2294 CCATCAGGGACTTTCGAGTCCGG aaatagaattggcgcgccttattaGCTTACAAAGTCTTTTAACCGCGG ACTCACCGCGGTTAAAAGAC >Rev_LCrescue 2295aaatagaattggcgc

TABLE C-6 Oligonucleotide primers used for generation of Omnitarg heavychain V_(H) + CH₁ by overlap extension SEQ ID SEQ ID For NO RevNO >For_HCrescue 2296 >Rev_HC1 2309 gtgctaataaggcgcAAAGGTACCACTAAAGGRATTGCGAA TAATAATTTTTTCACTATGACTGT >For_HC12297 >Rev_HC2_OT 2310 gtgctaataaggcgcgccaattctatACGCAGAGAACCGCCTGGCTGCACCA ttcaaggagacagtcatagtgaaaGCCCACCTCCGCTTTCCACCAGCT >For_HC2_OT 2298 >Rev_HC3_OT 2311tttagtggtacctttctattctcact TTTCACGTTCACTGATTATACCATGGccGAGGTTCAGCTGGTGGAAAGCG ATTGGGTTCGCCAGGCGCCGGGTA >For_HC3_OT2299 >Rev_HC4_OT 2312 GGCGGTTCTCTGCGTCTGAGCTGCGCCCCTTAAAACGTTGGTTGTAAATTGA TGCCTCCGGTTTCACGTTCACTGAGCCACCAGAGTTAGGGTTTACGTC >For_HC4_OT 2300 >Rev_HC5_OT 2313GCCAGGCGCCGGGTAAAGGCCTGAA TTCTGCACCCAGCGAATTCATCTGTATGGGTGGCCGACGTAAACCCTAAC AATAGAGTGTGTTTTTAGAGCGAT >For_HC5_OT2301 >Rev_HC6_OT 2314 CCAACGTTTTAAGGGTCGTTTCACCCTGCCTTGGCCCCAATAGTCAAAGTAA TGAGCGTAGATCGCTCTAAAAACAAAGGACGGGCCCAGATTGCGTGCA >For_HC6_OT 2302 >Rev_HC7 2315TCGCTGCGTGCAGAAGACACCGCTGT GATTTCGAGCTTGGGGCCAGCGGAAATTATTACTGTGCACGCAATCTGGG CACTGACGGACCTTTAGTGCTTGC >For_HC7_OT2303 >Rev_HCrescue_VH_OT 2316 ATTGGGGCCAAGGCACGTTGGTCACC GATTTCGAGCTTGGGGTGAGTAGCGCAAGCACTAAAGGT >Rev_HC7_OT_PCR 2304 >Rev_HC8 2317ACCTTTAGTGCTTGCGCTACTCACGG GGAGACAGTGACCGGTTCCGGGAAGTTGACCAACGTGCCTTGGCCCCAAT AATCTTTCACCAGACAGCCCAGCG >For_HC8 2305 >Rev_HC92318 CCCAAGCTCGAAATCCACGTCCGGTG TATACAAGCCGCTAGACTGCAAAACCGCACCGCCGCGCTGGGCTGTCTGG GCAGGGAAAGTATGTACACCCGAG >For_HC92306 >Rev_HC10 2319 CCGGTCACTGTCTCCTGGAACTCGGGTGGTTCACATTGCAAATATACGTCTG TGCACTTACCTCGGGTGTACATACGGTGCCCAGAGAGCTTGAAGGCAC >For_HC10 2307 >Rev_HC11 2320CTAGCGGCTTGTATAGCCTGTCAAGC TTTTTGTTCTGCGGCCGCACAGCTCTGTTGTGACCGTGCCTTCAAGCTCT TCGGTTCCACTTTCTTATCCA >For_HC112308 >Rev_HCrescue 2321 TTGCAATGTGAACCACAAACCGAGTA TTTTTGTTCTGCGGCACACCAAAGTGGATAAGAAAGTGG

1. A method for obtaining a polypeptide construct directed against toone or more antigens and/or epitopes and having one or more desiredcharacteristics, wherein said polypeptide construct essentially consistsor comprises at least two single domain antibodies, said method at leastcomprising the steps of: (i) selecting a template polypeptide constructdirected against one or more antigens or epitopes, (ii) producing adiversity of polypeptide constructs that are structural variants of theselected template polypeptide construct of step (i), wherein saidstructural variants each comprise at least two single domain antibodies,and (iii) screening the produced diversity of polypeptide constructs ofstep (ii) for a polypeptide construct having said one or more desiredcharacteristics, wherein said polypeptide construct comprises at leasttwo single domain antibodies and is directed against one or moreantigens and/or epitopes.
 2. The method according to claim 1, whereinsaid diversity of polypeptide constructs is a library of polypeptideconstructs.
 3. The method according to claim 1, wherein said structuralvariants of step (ii) are one or more of the following: structuralvariants with regard to the number and/or identity of said single domainantibodies in said polypeptide constructs, and/or, structural variantswith regard to the relative position of said single domain antibodieswithin the polypeptide constructs, and/or, structural variants withregard to the amino acid residues of the CDR region(s) of one or more ofsaid single domain antibodies in said polypeptide constructs, and/or,structural variants with regard to the amino acid residues of theframework region(s) of one or more of said single domain antibodies insaid polypeptide constructs, and/or, structural variants with regard tothe codon usage in the nucleic acid sequences encoding the polypeptideconstructs.
 4. The method according to claim 1, wherein said structuralvariants of step (ii) comprise at least two single domain antibodiesthat are linked via one or more peptide linkers.
 5. The method accordingto claim 4, wherein said structural variants of step ii) are one or moreof the following structural variants with regard to the composition ofsaid one or more peptide linkers, and/or, structural variants withregard to the number of said one or more linkers, and/or, structuralvariants with regard to the relative position of said one or morelinkers in said polypeptide constructs.
 6. The method according to claim1, wherein the single domain antibodies present in said diversity ofpolypeptide constructs and in said polypeptide construct are all heavychain variable domains or are all light chain variable domains.
 7. Themethod according to claim 6, wherein the single domain antibodiespresent in said diversity of polypeptide constructs and in saidpolypeptide construct are all heavy chain variable domains.
 8. Themethod according to claim 7, wherein the heavy chain variable domainsare variable domains obtainable from heavy chain antibodies (VHH). 9.The method according to claim 1, wherein in step (iii) the produceddiversity of polypeptide constructs is screened for a polypeptideconstruct having a suitable binding affinity or avidity, for apolypeptide construct having a suitable solubility, for a polypeptideconstruct having a suitable stability, for a polypeptide constructhaving a suitable efficacy, and/or for a polypeptide construct having asuitable potency. 10.-13. (canceled)
 14. The method according to claim1, wherein said diversity of polypeptide construct comprises at leasttwo single domain antibodies that are directed against the sameepitopes.
 15. The method according to claim 1, wherein said diversity ofpolypeptide construct comprises at least two single domain antibodiesthat are directed against at least two different epitopes that arepresent on the same antigen.
 16. The method according to claim 1,wherein said diversity of polypeptide construct comprises at least twosingle domain antibodies that are directed against at least twodifferent epitopes that are present on different antigens.
 17. Themethod according to claim 1, wherein said polypeptide constructcomprises at least three single domain antibodies.
 18. The methodaccording to claim 17, wherein said three single domain antibodies aredirected against the same epitope.
 19. The method according to claim 17,wherein said three single domain antibodies are directed against atleast three different epitopes.
 20. The method according to claim 19,wherein said at least three different epitopes are present on the sameantigen.
 21. The method according to claim 19, wherein said at leastthree different epitopes are present on at least two different antigens.22. Polypeptide construct obtainable by the method of claim 1.